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Gas Chromatographic Analysis of Sulfur Mustard in

Diethyl Phthalate

Paul A. Lancaster

Combatant Protection and Nutrition Branch


Aeronautical and Maritime Research Laboratory

DSTO-TR-0703

ABSTRACT

A gas chromatographic method for the analysis of 2,2'-dichlorodiethyl sulfide


(commonly known as Sulfur Mustard) that had been trapped in the solvent, diethyl
phthalate is described. The method utilises the improved sensitivity and selectivity
offered by the new Pulsed Flame Photometric Detector to detect routinely samples
containing 0.25 µg/mL of sulfur mustard in diethyl phthalate. The method is capable
of fast and reliable analysis of samples containing 0.25-5.0 µg/mL of sulfur mustard in
diethyl phthalate. Quality control measures determined that the method calibration
was still within 5% control limits after nearly 500 sample injections.

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Approved for public release

D E P A R T M E N T O F D E F E N C E
——————————!———————————
DEFENCE SCIENCE AND TECHNOLOGY ORGANISATION
Published by

DSTO Aeronautical and Maritime Research Laboratory


PO Box 4331
Melbourne Victoria 3001 Australia

Telephone: (03) 9626 7000


Fax: (03) 9626 7999
© Commonwealth of Australia 1998
AR-010-603
August 1998

APPROVED FOR PUBLIC RELEASE


Gas Chromatographic Analysis of Sulfur
Mustard in Diethyl Phthalate

Executive Summary
The aim of the project was to develop a gas chromatographic method to analyse
samples of the chemical warfare blister agent 2,2’-dichlorodiethyl sulfide (commonly
known as Sulfur Mustard or HD), that had been trapped in the solvent diethyl
phthalate (DEP). The samples were generated from chemical penetration experiments
performed on protective clothing.

The technique involved:


i. developing a gas chromatographic method that was capable of both meeting the
required detection limits and completing the analysis in a minimal time,
ii. preparation of standards that are representative of the samples to be analysed,
iii. establishing a quality control procedure to confirm the integrity of the method.

It was decided that, due to time constraints, the lowest possible detection limit would
not be an aim of this project. Instead a value of 0.5µg/mL of sulfur mustard in DEP
was set as the lowest probable concentration that would be required to be detected
routinely in the protective clothing penetration experiments. In order to prove that the
GC method could routinely detect such levels, the concentration was halved and the
ability to detect 0.25 µg/mL samples of sulfur mustard in DEP (or 0.35 ng of sulfur
mustard injected on column) was set as the final goal. Improved detection of sulfur
mustard was achieved by utilising a new design of gas chromatography detector
(Pulsed Flame Photometric Detector) which offers increased selectivity for sulfur (and
phosphorous) detection and improvements in sensitivity of up to 2 orders of
magnitude above that of a normal flame photometric detector. The method developed
was found to not only meet the stated sensitivity requirements, but also exhibit
excellent reproducibility. Quality control measures established that the method
calibration stayed within the 5% deviation control limits after analysis of almost 500
samples.
Authors

Paul A. Lancaster
Combatant, Protection and Nutrition Branch

Paul Lancaster, BAppSci (App Chem) is a Professional Officer at


AMRL-Maribrynong. Before joining DSTO in 1994, he worked
at La Trobe University School of Agriculture investigating the
fertility status of crop and pasture soils. His work in DSTO
includes the development of procedures for using detection and
monitoring equipment for chemical warfare agents, bioaerosols
and other hazardous vapours.
____________________ ________________________________________________
Contents

1. INTRODUCTION .............................................................................................................. 1

2. INSTRUMENTATION ...................................................................................................... 2
2.1 Pulsed Flame Photometric Detector (PFPD) .................................................................. 2
2.1.1 The flame pulsation cycle........................................................................................ 3
2.1.1.1 Gas flows/composition and chamber fill times ............................................ 3
2.1.2 Emission profiles ...................................................................................................... 4
2.1.3 Detector Sensitivity .................................................................................................. 4

3. EXPERIMENTAL PROCEDURES ................................................................................... 5


3.1 Gas Chromatography Instrumentation and Conditions ............................................. 5
3.1.1 Varian 3400 GC Set Up. ........................................................................................... 5
3.1.2 Pulsed Flame Photometric Detector. ..................................................................... 5
3.1.2.1 Optimisation of PFPD. ...................................................................................... 6
3.1.3 Varian 8200 CX Auto Sampler................................................................................ 6
3.1.4 Star Chromatography Workstation. ...................................................................... 6
3.2 Quantification/ Preparation of Standards...................................................................... 7

4. RESULTS AND DISCUSSION........................................................................................ 8

5. CONCLUSION.................................................................................................................. 10

6. REFERENCES .................................................................................................................... 11

7. ACKNOWLEDGMENTS ................................................................................................ 11

8. GLOSSARY OF ACRONYMS........................................................................................ 11

9. TABLES............................................................................................................................... 12

10. FIGURES............................................................................................................................. 14

APPENDIX 1: MSDS FOR DISTILLED MUSTARD (HD)............................................ 19

APPENDIX 2: CPNB VARIAN 3400/PFPD START UP PROCEDURE........................ 25

APPENDIX 3: PFPD MASS FLOW CONTROLLER CALIBRATION ......................... 27

APPENDIX 4: STAR CHROMATOGRAPHY WORKSTATION SETUP................... 28

APPENDIX 5: PURITY DETERMINATION OF SULFUR MUSTARD BY GC-MS . 29


DSTO-TR-0703

1. Introduction
In order to monitor samples of the chemical warfare blister agent 2,2’-dichlorodiethyl
sulfide (or sulfur mustard), trapped in diethyl phthalate as a result of experiments
performed on protective clothing, an improved analytical procedure which provided
appropriate sensitivity in a realistic time was sought.

Although being clear and odourless when pure, sulfur mustard is normally an amber
to black coloured liquid with a characteristic smell similar to garlic or rotten onions.
At room temperature, mustard agent is a liquid with low volatility (vapour pressure
0.11 mm Hg @ 25°C) and is very stable during storage. Sulfur mustard is soluble in
fats and oils and can easily be dissolved in most organic solvents but has negligible
solubility in water. In alkali solutions, sulfur mustard decomposes (hydrolyses) into
non-poisonous products. As sulfur mustard forms large globules that are suspended
in the alkali solution, with only the surface layer in contact with the alkali solution, the
decomposition process proceeds slowly. Usually sulfur mustard is disposed of by
leaving it in a hypochlorite solution for approximately 24 hours (see appendix 1 for
disposal information).

The technique chosen for the detection of sulfur mustard involved a new design of gas
chromatography (GC) detector (Pulsed Flame Photometric Detector - PFPD), that has
been claimed to provide increased selectivity and sensitivity to sulfur detection. The
study involved developing a GC method capable of performing the analysis in a
minimal time, preparation of standards that are representative of the samples to be
analysed and establishment of a quality control procedure to confirm the integrity of
the analysis.

Due to time constraints, attainment of the lowest possible detection limit was not
attempted. Instead a value of 0.5µg/mL of sulfur mustard in DEP was set as the
lowest probable concentration of sulfur mustard to be encountered in protective
clothing penetration experiments. To demonstrate that this level could reliably be
detected using this technique, this concentration was halved and the ability to
routinely detect samples containing 0.25 µg/mL of sulfur mustard in DEP was defined
as a goal.

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2. Instrumentation
The sulfur mustard samples were analysed using a Varian 3400 Gas Chromatograph.
The samples were introduced to the gas chromatograph using a Varian 8200-CX auto
sampler and, after separation had occurred, detected using a Varian Pulsed Flame
Photometric Detector (PFPD see 2.1). The detector signal was processed using the Star
chromatography workstation (ver 4.51) data handling system. The main advantages of
using the PFPD include improved detection sensitivity (approximately 2 orders of
magnitude for S2) for sulfur and phosphorus, increased detection selectivity against
hydrocarbon molecules (approximately 3 orders of magnitude in the sulfur mode),
lower gas consumption, reduced emission quenching, additional temporal information
and the ability to detect selectively other heteroatoms such as nitrogen, or the
simultaneous detection of sulfur and carbon.1

2.1 Pulsed Flame Photometric Detector (PFPD)


The Pulsed Flame Photometric Detector is a development of the Flame Photometric
Detector (FPD) that has long been the detector preferred for the selective measurement
of sulfur and phosphorus compounds. The PFPD is a new design which employs a
pulsed-flame operation instead of the continuous-flame mode of detection. The
pulsed-flame mode is based on a flame source and combustible gas flow that cannot be
sustained. The combustible gases (H2 and air) are mixed in a small combustion
chamber before entering an ignition chamber that contains a continuously-heated
igniter coil. The ignited flame then propagates back to the combustible mixture source
and is self - terminated after the combustible gas mixture is consumed. The
continuous gas flow then exhausts the combustion products and refills the ignition
chamber creating an additional ignition when the mixture reaches the igniter coil. This
process continues in a periodic fashion at a rate of about 2-4 Hz.

Due to the pulsing nature of the PFPD the processing electronics have been modified
to work with pulsed signals. The derived emissions appear as discrete signals
separated in time. These discrete signals are stored for each pulse and used to generate
a peak (waveform). While this peak contains all the signal information from the
chromatographic peak it may or may not have an identical shape. Given this,
quantification is normally based on peak areas which are less affected than peak
heights by the pulsing nature of the detector and the processing electronics of the
emission signal.

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2.1.1 The flame pulsation cycle.

This cycle involves four stages (Figure 1):


1. Fill: The air (Air 1) and hydrogen are mixed, before entering the combustor
chamber through the interior (combining with the column effluent) and around the
outside of the quartz combustor tube. A split valve is employed to set the ratio of
air to hydrogen flow through and around the quartz combustor tube. Additional
air flow (Air 2) into the wall flow provides a means of adjusting the ratio of air to
hydrogen and balancing the fill rate of the combustor and igniter chambers.

2. Ignition: The igniter chamber contains a continuously heated Ni/Cr wire igniter.
When the combustible mixture makes contact with this coil ignition occurs.

3. Propagation: The ignited flame front then propagates downward to the gas source
in the combustor chamber. This chamber contains the column effluent. The flame
is self terminated once all of the combustible gas mixture present in the combustion
path is consumed. The combustion products are swept from the detector (a result of
the continuous gas flow through the detector) via a vent located at the top of the
igniter chamber. The fill stage then begins again and the process repeats itself.

4. Emission: During and after flame propagation, light is emitted by the excited
molecules that result from the combustion of the column effluent. To gain
maximum sensitivity this must occur in the combustor chamber. The flame
background emits light for less than 0.3 milliseconds after propagation, whereas
sulfur (and phosphorus) containing molecules will emit light over a longer period.
It is this difference that adds to the selectivity and sensitivity of the PFPD.

2.1.1.1 Gas flows/composition and chamber fill times

The flow paths of gases entering and exiting the detector are shown in Figure 2. The
sensitivity and selectivity of the PFPD are dramatically affected by the relative flow
rates of these gases. The flame pulsation rate is determined by the total flow of air and
hydrogen entering the detector. When adjusting the gas mixture composition it is
important to strike a balance between the time required to fill the combustor chamber
and the time required to fill the igniter chamber. If the combustor chamber does not
fill slightly before the igniter chamber then the analyte response will be markedly
decreased due to the pulsed flame only propagating into the combustor on every other
pulse. This results in incomplete combustion of the sample before it vents from the
detector and greatly increased noise due to ignition occurring on alternate pulses
(referred to as tick-tock mode). In the opposite situation, if the combustor chamber
fills too quickly (relative to the igniter chamber), again a reduction in analyte response
will result. This is due to a portion of the sample exiting the combustor chamber
before complete combustion of the column effluent has occurred. The fill times of the
two chambers can be adjusted by the air/hydrogen flow entering the detector.

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2.1.2 Emission profiles

The composition of the combustible gas mixture is a determining factor in the


character and intensity of the flame emission. Hydrogen rich flames operate at a lower
temperature that favours gas phase chemical reactions with products that emit light
(background emission) in the blue region. Hydrocarbon-related emission, however, is
very short and its time scale is shorter than the time of pulsed-flame front propagation
through the viewing zone.1 In contrast, products from sulfur containing species (S2)
form later and have greater lifetimes than background species. Figure 3 shows the
time-dependant separation of the hydrocarbon and sulfur emission profiles. It can be
seen that the OH, CH and C2 emissions in a hydrogen-rich flame occur and then decay
within 4 milliseconds. From these emission profiles it is obvious that S2 emission is
negligible during OH emission and that the S2 emission reaches a maximum about 5-6
milliseconds after the OH emission and lasts 2-6 times longer than the emission of the
species responsible for background emission. This phenomenon allows the easy
separation of the sulfur emission from any residual hydrocarbon emissions. This time
dependent separation is achieved by the use of a simple electronic amplifier that
integrates the emission related current within a delayed time gate window. By tuning
the gate delay (the period between flame ignition and the start of integration of the
emission) and gate width (the period over which integration occurs) to the needs of
specific analysis, considerable improvements in the sensitivity and selectivity of sulfur
and phosphorous containing species are possible.

2.1.3 Detector Sensitivity

The sensitivity of the PFPD is reliant upon the emission duration across the viewing
window of the photomultiplier tube.1 The emission duration is dependent on the
opening diameter of the viewing windows and the flame velocity. In general,
increasing detector temperature increases the flame velocity, resulting in a decrease in
the emission duration and a reduction in the sensitivity of the detector. There are
three main reasons why the PFPD has a reported two orders of magnitude increase in
sensitivity:

• Noise from the flame background and the photomultiplier tube is


significantly reduced by the delayed gated integration of the emission profile.
• Increased brightness of the flame results from reduced total gases and a
smaller flame volume. This results in an increase in the sulfur signal which is
dependent on the square of the sulfur concentration. For this reason a narrow
bore quartz combustor tube is used to increase sensitivity for sulfur analysis.
• The increased selectivity allows the narrow band interference filter to be
replaced with the cheaper and more stable1 wide band coloured glass filter.
This results in an increase in the heteroatom signal by one order of
magnitude. Although the carbon response is significantly increased, this
effect is removed by the time-delayed gate integration.

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3. Experimental Procedures

3.1 Gas Chromatography Instrumentation and Conditions


A Varian 3400 GC with a Varian 8200-CX auto sampler and Star chromatography
workstation (ver 4.51) data handling system was used for the analysis. The gas
chromatograph contained the Varian Pulsed Flame Photometric Detector (see 2.1). The
instrument start up procedure is described in appendix 2.

3.1.1 Varian 3400 GC Set Up.

The Varian 3400 GC was set up as specified in Table 1. Samples were injected using a
Varian 1041 universal injector with a capillary column adaptor fitted. A non-polar
SGE 12QC5/BP1 (see Table 2) fused silica capillary column was used to separate the
samples. The carrier gas was high purity nitrogen (BOC Gases Australia) used at a
flow rate of 3.0 mL/min.

The gas chromatograph temperature settings were as follows:


• Injector 275ºC
• Oven 230ºC
• Detector 250ºC

Chromatograms (GC runs) were 0.65 minutes in length with the sulfur mustard peak
having a retention time of approximately 0.4 minutes (see Figure 4). A two minute
equilibration time was programmed between each run.

3.1.2 Pulsed Flame Photometric Detector.

The PFPD has a large number of variables that all require optimisation (and re-
optimisation) in order to achieve improved sensitivity. As changing one gas flow may
alter the balance of the detector chambers filling rate, the optimisation process has no
regimented procedure except to employ a systematic approach of trial and error
adjustments. Firstly, the dial indicators on the flow controllers must be calibrated,
noting that the flow controllers must have pressure applied for 24 hours before
constant flows are possible. This is achieved by closing all cylinders and flow
controllers, then sequentially turning on the cylinder and increasing the flow controller
by increments of 100 units while noting the flow exiting the detector. This procedure
is carried out for all flow controllers (for calibration curves see appendix 3). For
optimum sulfur sensitivity it is recommend that the narrow bore quartz combustor is
used along with a detector temperature of 200 °C2, although after optimisation this was
set at 250°C.

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DSTO-TR-0703

3.1.2.1 Optimisation of PFPD.


The flow controllers were set to the suggested gas flow2 and the split valve adjusted so
that the detector was just out of “tick-tock” mode. The described procedure, however,
did not achieve the expected detector response. This was overcome by first turning the
split valve fully clockwise, and then turning it anti-clockwise (noting the number of
turns) until the irregular “tick-tock” pulse became regular again. The split valve was
adjusted ¼ turn anti-clockwise/clockwise and a standard sample injected noting the
sulfur response. This procedure was repeated until a maximum sulfur response was
achieved. The response of the PFPD increases when hydrogen rich conditions are
used. The H2 flow controller was therefore increased by ten units and then the
standard solution was injected 3 times to determine sulfur response (ie peak area). The
H2 flow is then increased (or decrease) until the sulfur response no longer increases. If
the pulse rate exceeds 4 pulses per second then the Air1 flow should be reduced by 10
units while continuing to increase the H2 flow. This adjustment should be repeated for
the Air1 and Air2 flow controllers, injecting a standard after each adjustment to
determine sulfur response. Once the Sulfur emission has been optimised the split
valve must again be adjusted such that the detector is just out of “tick-tock” (see
above). Finally the detector temperature should be adjusted in a similar manner to
that of the flow controllers (ie increase/decrease detector temperature by 20°C then
inject standard to determine Sulfur response). Optimum PFPD configuration for this
method is detailed in Table 3.

3.1.3 Varian 8200 CX Auto Sampler.

The 8200 autosampler is a pneumatic/motor driven autosampler that allows the


automated analysis of up to 48 samples. The sampler is mounted directly over the
injector on the top cover of the 3400 GC. Samples may be injected using the preset
modes or, as in the case of this method, a user defined mode.
Programmable parameters include:
• Injection volume (0.1-5.0µL) and rate (0.2-10µL/sec)
• Sample uptake rate
• Syringe needle depth and solvent wash cycle
• Residence time and hot needle time
The settings of the 8200 autosampler used in this method are listed in Table 4. The
optimised injection volume of 1.4 µL was determined by injecting increasing volumes
of a known standard and observing the detector response. A similar method of
optimisation was used for determining the injection rate.

3.1.4 Star Chromatography Workstation.

The Star Chromatography workstation, Version 4.51 (see Appendix 4 for method
setup), is a software package for automated data acquisition, analysis, and reporting of
samples analysed on Varian GC instruments. The system receives the detector output,
storing the raw data on a Laboratory PC's hard disk before performing arithmetical
manipulations and producing a detailed report page for individual injections.

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3.2 Quantification/ Preparation of Standards.


Quantification was accomplished by preparing standard solutions of sulfur mustard in
purified diethyl phthalate with a range of 0.25 to 5.0 µg/mL. DEP was chosen as the
solvent as this is the solvent commonly used to trap sulfur mustard vapour in
experiments. Analytical standards were made from pure sulfur mustard diluted to
100mL with DEP. In previous experiments this sample of sulfur mustard had been
found to be 99.9% pure. To determine if significant degredation had occured since this
analysis had been performed, the sulfur mustard was dissolved in dichloromethane
and analysed on a gas chromatograph linked to a mass spectrometer (GC-MS). The
resulting total ion chromatogram indicated that the sulfur mustard sample was greater
than 99% pure (see appendix 5). This stock solution of sulfur mustard was then used
to make up a solution of DEP containing 50 µg/mL sulfur mustard, which was then
diluted to make the working solutions (see Table 5). Experiments determined that
these working solutions were valid for periods of up to 12 weeks. A period of 6 weeks
was set as the maximium time that standards were to be used before replacement.
Analytical working curves, that plot concentration of sulfur mustard standards
(µg/mL) verses the peak area counts of the identified sulfur mustard peak, were
generated by the Star Chromatography system. Figure 5 shows the chromatograms
typical of the standards that were used to generate such calibration curves. The
instrument calibration was broken into standards of low and high concentrations of
sulfur mustard. The high concentration standard calibration curve (1.0-5.0µg/mL) was
a cubic curve fit that forced the origin through zero (see Figure 6) and commonly had a
correlation coefficient greater than R2=0.995. The low level standard calibration curve
(0.25-1.0µg/mL) was a linear curve fit that included the origin (see Figure 7). Again
the correlation coefficient was commonly greater than R2=0.995.

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4. Results and Discussion


The analytical method required many months of development before the aims of the
project were achieved. The long development period can be attributed to two main
reasons:

Firstly, there were a number of problems with the hardware of the GC system. Before
attempting any method optimisation the GC components were completely overhauled.
The injector and detector were thoroughly cleaned and the column was refitted and
any leaks repaired. The peak areas of consecutive injections of the same sample
showed a high degree of variation. This problem was traced to a faulty syringe in the
autosampler, which was subsequently replaced, resulting in a marked improvement in
reproducibility. A dramatic lack of sensitivity was significantly improved by
increasing the sample injection rate of the autosampler from 1 to 2µL/second. These
repairs and changes resulted in a 10 fold increase in sulfur mustard peak areas. It
should be noted that with a detector as complicated as the PFPD, it can be very
difficult to troubleshoot during method development. It can be very time consuming
trying to optimise the detector when the problem resides in other components of the
GC system. It is also very difficult to track down faults with other system components
without first optimising the detector, albeit it crudely.

The second issue was that the detector response did not follow that described in the
PFPD Operators Manual. The manual suggests a detector operating temperature of
200°C and indicates that increasing this temperature will result in a decreased sulfur
response2. While this may be true it was found that at lower temperatures, the peak
areas did increase, but, the peaks also broadened and distorted so badly that poor
reproducibility resulted. A detector temperature of 250°C was found to provide the
best compromise between sensitivity and reproducibility. The manual also
recommends use of a hydrogen rich flame, however it was found that this resulted in
broad sulfur mustard peaks that exhibited poor sensitivity. A leaner flame gave the
best compromise between high sensitivity and acceptable reproducibility. It was also
found that baseline observations made while adjusting the Air-H2 needle valve (to
establish tick-tock - see 2.1.2) did not correspond to those described.2 As a
consequence the optimum setting of the Air-H2 needle valve was determined by
performing multiple injections of a standard sulfur mustard solution.

As DEP is one of the more viscous solvents that a chromatographer will encounter, a
long and complicated syringe filling regime is required to present a representative
sample that contains no air bubbles to the injector. This regime increased the analysis
time considerably. The syringe fill time, approximately 5 minutes, put tight limitations
on the length of each chromatographic run. If a reasonable sample throughput was to
be maintained, the sample analysis would have to be as short as possible. For this
reason a short (12 meter) non-polar fused silica column was selected and was found to
provide excellent separation in less than one minute analysis time. To aid the injection
of larger volumes of the viscous sample on to the chromatographic column a megabore

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column (id=0.53mm) was the preferred option. This decision was made with full
knowledge that approximately one order of magnitude in sensitivity would be
sacrificed as a direct result of the increased column flow.1 It was hoped that this loss in
sensitivity would be compensated for by taking advantage of the ability of the PFPD to
cope with large amounts of solvent (ie greater sample volumes). As the sulfur
response is proportional to the square of the sulfur concentration it was considered
that larger sample volumes would aid in the trace level detection of sulfur in DEP.

Previous flame photometric methods used to analyse samples of sulfur mustard in


DEP had detection limits of 0.5µg/mL. One of the aims of this method development
using the PFPD was to be able to routinely analyse samples that contain less than
0.5µg/mL of sulfur mustard. This was achieved using the described method with
standards that contain 0.25µg/mL of sulfur mustard being analysed on a routine basis.
This sensitivity was achieved by optimising only the basic variables of the PFPD (ie
flow rates, split valve, detector temperature, combustor tube).
Greater sensitivity, however, should be achievable by optimising parameters such as:
• Photomultiplier tube voltage
• Gate delay
• Gate width
• Gain
At present, greater improvements in the sensitivity of the method are not considered
necessary, as the detection requirements have already been met.

To provide an indication of the robustness of the method, the ability to maintain


calibration levels within preset limits was investigated. The low level method was
calibrated, then used to analyse a large number of samples that had been generated
from clothing penetration tests. All samples were injected four times as standard
practice, then the 0.975 µg/mL sulfur mustard standard, used to calibrate the method,
was injected in triplicate every 8 samples, or 32 injections. This procedure was
repeated for a total of 490 injections and carried out over a period of 17 days. The
resulting control chart (Figure 8) shows that of the 42 times that the verification
standard was injected (during the 490 total injections) the deviation of the calculated
concentration, from the known concentration of the standard, exceeded the 5% control
limit only 5 times. Furthermore, all injections that fell outside the control limit were
still within 6% of the known concentration of the standard. The control chart also
shows that at no time did the average value for the verification standard (the dashed
line in the control chart ) fall outside the 5% control limits. This indicates that the
calibration curve will remain valid for at least two weeks or 500 injections. This is
consistent with the work load that the system would be expected to undertake during
normal operations without requiring maintenance. For the method development,
stringent control limits of 5% were considered appropriate.

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For quality control during routine analysis a standard sample would be injected in
triplicate every 8 samples (or 32 injections) to verify the integrity of the calibration
curve. It was decided that a 10% control limit was suitable as the alarm point that
would alert the operator to recalibrate the method. This alarm can be pre-set in the
verification setup of the Star Chromatography Workstation, by setting the deviation
tolerance to 10%. When verification injections fall between the 5 and 10% control
limits, results should be viewed critically and the method calibration carefully
monitored.

5. Conclusion
A gas chromatographic procedure has been developed that analyses samples of DEP
containing varying amounts of the chemical warfare agent sulfur mustard. These
samples are typical of those generated during clothing penetration studies. The
procedure utilises a new design of GC detector based on a pulsed version of the
commonly used flame photometric detector. The system provides the selectivity and
sensitivity required to fulfil the goal of routine analysis of samples containing 0.25
µg/mL of sulfur mustard in DEP. This concentration equates to 0.35 ng of sulfur
mustard injected on to the GC column. Although the initial set up and detector
optimisation were found to be time consuming, once running, the detector requires
minimal maintenance.

The procedure was found to be robust with the method calibration able to be
maintained within a stringent 5% error tolerance for long periods. It is recommended
that the method be recalibrated every 2 weeks or 500 sample injections, which ever
comes first. It is further recommended that a quality control program be introduced
into the analysis. This would involve injecting (in triplicate) a standard sample every
32 injections. If any of the three standard injections fall outside 10% control limits the
method should be recalibrated immediately. If two or more of the injections should
fall between the 5% and 10% control limits then the results should be critically
analysed.

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6. References

1 Cheskis S, Atar E & Amirav A. Pulsed-Flame Photometer: A Novel Gas


Chromatography Detector. Anal. Chem. 65, 539-555, 1993.
2 Varian Chromatography Systems. Pulsed Flame Photometric Detector: - Operators
Manual. Varian Associates, Inc. 1995.

7. Acknowledgments
Eric Mattsson for performing GC-MS analysis to determine the purity of the sulfur
mustard used to make up standard solutions.

Bernie Gray for providing samples of sulfur mustard that had been generated during
clothing trials performed in the Combatant Protection & Nutrition Branch.

Denys Amos for providing editorial advice in the preparation of this report.

8. Glossary of Acronyms
CPNB Combatant Protection and Nutrition Branch
DEP diethyl phthalate
DSTO The Defence Science and Technology Organisation
FPD Flame Photometric Detector
GC gas chromatography
GC/MS gas chromatography/mass spectrometer
HD Sulfur Mustard: 2,2'-dichlorodiethyl sulphide
Hz hertz
kPa kilopascal
mL millilitres
ng nanograms
PFPD Pulsed Flame Photometric Detector
µg micrograms
µL microlitres

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9. Tables

Table 1. Setup for Varian 3400 GC.


Column: SGE 12QC5/BP1 1.0
Oven: 230°C, isocratic (for 0.65 minutes) run time
with a 2 minute stabilisation time between injections
Injector: 1041, 275°C
Detector: Varian PFPD, 250°C (Att=1; Range=10)
Auto Sampler: Varian 8200CX
Data System: Varian Star Workstation ver 4.51
Carrier: Nitrogen, 3.0 mL/min
Injections: 1.4 µL of sample at a rate of 2.0 µL/sec.

Table 2. Column Specifications for: SGE 12QC5/BP1 1.0.


Length 12 Meter
Film Thickness 1.0 µm
Internal Diameter 0.53 mm
Outside Diameter 0.68 mm
Type Bonded Phase
Material Fused Silica
Phase Dimethyl siloxane (non-polar)
Operating Temperatures -60 to 315 ºC

Table 3. Setup for PFPD.


H2: 11.87 mL/min (Dial Setting=775a)
Air-1: 14.76 mL/min (Dial Setting=630a)
Air-2: 10.02 mL/min (Dial Setting=450a)
Quartz Combustor tube Narrow bore
Attenuation 1
Range 10
Full Scale 10 Volts
Bunch Rate 8 Points (5.0 Hz)
Noise Monitor Length 64 Bunched Points (12.8 seconds)
Photomultiplier tube high voltage 550 Volts
Pulse Rate 2-4 Hz
Gate Width 1.0 milliseconds
Gate Delay 1.0 milliseconds
Trigger Level 200 millivolts
Air1/H2 split valve 4.375 turns anti-clockwise
a for flow controller calibration charts see appendix 3

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Table 4. 8200 Autosampler Settings.


Mode User Defined
Syringe Wash Solvent Wash A (EtOH) then B( CH2Cl2)
Solvent Wash Time 10 seconds
Vial Needle Depth 90%
Uptake Speed 1.0 µL/sec
Pause Time 10 seconds
Inject Rate 2.0 µL/sec
Injection Volume 1.4 µL

Table 5. Preparation of Standard Solutions.


Mass of sulfur mustard Volume of DEP
Stock Solution " 1 drop (30-50mg) of HD 100 mL
50µg/mL Std Solution # appropriate mass of " 25 mL
5µg/mL Std Solution 2.5g of # 25 mL
4µg/mL Std Solution 2.0 of # 25 mL
3µg/mL Std Solution 1.5 of # 25 mL
2µg/mL Std Solution 1.0 of # 25 mL
1µg/mL Std Solution 0.5 of # 25 mL
0.5µg/mL Std Solution 0.25 of # 25 mL
0.25µg/mL Std Solution 0.125 of # 25 mL

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10. Figures

Figure 1: Stages of the Pulsed Flame Photometric Detector’s pulsed-flame operation2.

Figure 2: Schematic of the Pulsed Flame Photometric Detector2.

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Figure 3: Hydrocarbon and S2 flame emission profile, as a function of time1

Figure 4: Chromatogram of 0.25µg/mL Sulfur Mustard standard. Sulfur mustard peak


elutes at 0.402 minutes

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Figure 5: Overlay of chromatograms of the standards used to calibrate this method. Standards
ranged from 0.25-5.0µg/mL of Sulfur Mustard in diethyl phthalate.

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Figure 6: High concentration method calibration curve. (1.0-5.0 µg/mL HD in DEP)

Figure 7: Low concentration calibration curve. (0.0-1.0 µg/mL HD in DEP)

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Figure 8: Control Chart with control limit set at 5% deviation from the calculated value of the
standard used. In this case the 0.9749 µg/mL HD standard, used to calibrate the
method, was analysed (three injections) after every 32 sample injections. The
dashed line represents the average of the three injections. The calibration curve
was still found to be accurate (within the 5% control limits) after nearly 500
injections had been made.

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Appendix 1: MSDS for Distilled Mustard (HD)


Note: This is an abridged version of the full MSDS that can be found at:
http://www.apgea.army.mil/safety/msds/hd1.html

SECTION I - GENERAL INFORMATION


DATE: 22 September 1988
REVISED: 28 February 1996

MANUFACTURER'S ADDRESS:

U.S. ARMY CHEMICAL BIOLOGICAL DEFENSE COMMAND


EDGEWOOD RESEARCH DEVELOPMENT, AND ENGINEERING CENTER (ERDEC)

CAS REGISTRY NUMBERS: 505-60-2, 39472-40-7, 68157-62-0

CHEMICAL NAME: bis-(2-chloroethyl)sulfide

TRADE NAMES AND SYNONYMS:

Sulfide, bis (2-chloroethyl)


Bis(beta-chloroethyl)sulfide
1,1'-thiobis(2-chloroethane)
1-chloro-2(beta-chloroethylthio)ethane
Beta, beta'-dichlorodiethyl sulfide
2,2'dichlorodiethyl sulfide
Di-2-chloroethyl sulfide
Beta, beta'-dichloroethyl sulfide
2,2'-dichloroethyl sulfide
H; HD; HS
Iprit
Kampstoff "Lost"; Lost
Mustard Gas
S-Lost; S-yperite; Schewefel-lost
Sulfur mustard; Sulphur mustard gas
Yellow Cross Liquid
Yperite

CHEMICAL FAMILY: Chlorinated sulfur compound

FORMULA/CHEMICAL STRUCTURE: C4H8Cl2S

ClCH2CH2-S-CH2CH2Cl

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SECTION II - HAZARDOUS INGREDIENTS


INGREDIENTS FORMULA PERCENTAGE BY AIRBORNE
NAME WEIGHT EXPOSURE LIMIT
(AEL)

Sulfur Mustard C4H8Cl2S 100 0.003 mg/m3

SECTION III - PHYSICAL DATA


BOILING POINT: 217 °C (422 °F)

VAPOR PRESSURE (mm Hg): 0.072 mm Hg @ 20 °C


0.11 mm Hg @ 25 °C

VAPOR DENSITY (AIR=1): 5.5

SOLUBILITY IN WATER: Negligible. Soluble in fats and oils, gasoline, kerosene,


acetone, carbon tetrachloride, alcohol, tetrachloroethane, ethylbenzoate, and ether.
Miscible with the organophosphorus nerve agents.

SPECIFIC GRAVITY (H2O=1): 1.27 @ 20 °C

FREEZING POINT: 14.45 °C

LIQUID DENSITY (g/cc): 1.268 @ 25 °C


1.27 @ 20 °C

PERCENTAGE VOLATILE BY VOLUME:


610 mg/m3 @ 20 °C
920 mg/m3 @ 25 °C

APPEARANCE AND ODOR:

Normally amber to black colored liquid with garlic or a horseradish odor. Water clear
if pure. The odor threshold for HD is 0.6 mg/m3 (.0006 mg/L).

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SECTION IV - FIRE AND EXPLOSION DATA

FLASHPOINT: 105 °C (Can be ignited by large explosive charges)

FLAMMABILITY LIMITS (% by volume): Unknown

EXTINGUISHING MEDIA: Water, fog, foam, CO2. Avoid use of extinguishing


methods that will cause splashing or spreading of HD.

SECTION V - HEALTH HAZARD DATA


AIRBORNE EXPOSURE LIMIT (AEL): The AEL for HD is 0.003 mg/m3 as found in
"AR 40-173, Occupational Health Guidelines for the Evaluation and Control of
Occupational Exposure to Mustard Agents H, HD, HT." To date, the Occupational
Safety and Health Administration (OSHA) has not promulgated a permissible
exposure concentration for HD.

EFFECTS OF OVEREXPOSURE: HD is a vesicant (causing blisters) and alkylating


agent producing cytotoxic action on the hematopoietic (blood-forming) tissues which
are especially sensitive. The rate of detoxification of HD in the body is very slow and
repeated exposures produce a cumulative effect. HD has been found to be a human
carcinogen by the International Agency for Research on Cancer (IARC).

Median doses of HD in man are:

LD50 (skin) = 100 mg/kg


ICt50 (skin) = 2000 mg-min/m3 at 21-27°C (70 - 80 °F - humid environment)
= 1000 mg-min/m3 at 32 °C (90 °F - dry environment)

ICt50 (eyes) = 200 mg-min/m3

ICt50 (inhalation) = 1500 mg-min/m3 (Ct unchanged with time)

LD50 (oral) = 0.7 mg/kg

Maximum safe Ct for skin and eyes are 5 and 2 mg-min/m3, respectively.

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ACUTE PHYSIOLOGICAL ACTION OF HD IS CLASSIFIED


AS LOCAL AND SYSTEMIC.

LOCAL ACTIONS: HD effects both the eyes and the skin. SKIN damage occurs after
percutaneous absorption. Being lipid soluble, HD can be absorbed into all organs.
Skin penetration is rapid without skin irritation. Swelling (blisters) and reddening
(erythema) of the skin occurs after a latency period of 4-24 hours following the
exposure, depending on degree of exposure and individual sensitivity. The skin
healing process is very slow. Tender skin, mucous membrane and perspiration-
covered skin are more sensitive to the effects of HD. HD's effect on the skin, however,
is less than on the eyes. Local action on the eyes produces severe necrotic damage and
loss of eyesight. Exposure of eyes to HD vapor or aerosol produces lacrimation,
photophobia, and inflammation of the conjunctiva and cornea.

SYSTEMIC ACTIONS: Occurs primarily through inhalation and ingestion. The HD


vapor or aerosol is less toxic to the skin or eyes than the liquid form. When inhaled,
the upper respiratory tract (nose, throat, tracheae) is inflamed after a few hours latency
period, accompanied by sneezing, coughing, and bronchitis, loss of appetite, diarrhea,
fever, and apathy. Exposure to nearly lethal doses of HD can produce injury to bone
marrow, lymph nodes, and spleen as showed by a drop in white blood cell count, thus
resulting in increased susceptibility to local and systemic infections. Ingestion of HD
will produce severe stomach pains, vomiting, and bloody stools after a 15-20 minute
latency period.

CHRONIC EXPOSURE: HD can cause sensitization, chronic lung impairment,


(cough, shortness of breath, chest pain), cancer of the mouth, throat, respiratory tract
and skin, and leukemia. It may also cause birth defects.

EMERGENCY AND FIRST AID PROCEDURES:

INHALATION: Hold breath until respiratory protective mask is donned. Remove


from the source IMMEDIATELY. If breathing is difficult, administer oxygen. If
breathing has stopped, give artificial respiration. Mouth-to-mouth resuscitation
should be used when approved mask-bag or oxygen delivery systems are not
available. Do not use mouth-to-mouth resuscitation when facial contamination exits.
Seek medical attention IMMEDIATELY.

EYE CONTACT: Speed in decontaminating the eyes is absolutely essential. Remove


the person from the liquid source, flush the eyes immediately with water for at least 15
minutes by tilting the head to the side, pulling the eyelids apart with the fingers and
pouring water slowly into the eyes. Do not cover eyes with bandages but, if necessary,
protect eyes by means of dark or opaque goggles. Transfer the patient to a medical
facility IMMEDIATELY.

SKIN CONTACT: Don respiratory protective mask. Remove the victim from agent
sources immediately. Immediately wash skin and clothes with 5% solution of sodium

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DSTO-TR-0703

hypochlorite or liquid household bleach within one minute. Cut and remove
contaminated clothing, flush contaminated skin area again with 5% sodium
hypochlorite solution, then wash contaminated skin area with soap and water. Seek
medical attention IMMEDIATELY.

INGESTION: Do not induce vomiting. Give victim milk to drink. Seek medical
attention IMMEDIATELY.

SECTION VI - REACTIVITY DATA


STABILITY: Stable at ambient temperatures. Decomposition temperature is 149 °C to
177 °C. Mustard is a persistent agent depending on pH and moisture, and has been
known to remain active for up to three years in soil.

INCOMPATIBILITY: Rapidly corrosive to brass @ 65 °C. Will corrode steel at a rate of


.0001 in. of steel per month @ 65 °C.

HAZARDOUS DECOMPOSITION: Mustard will hydrolyze to form HCl and


thiodiglycol.

HAZARDOUS POLYMERIZATION: Does not occur.

SECTION VII - SPILL, LEAK, AND DISPOSAL PROCEDURES


STEPS TO BE TAKEN IN CASE MATERIAL IS RELEASED OR SPILLED: If spills
or leaks occur, only personnel in full protective clothing will remain in the area. In
case of personnel contamination See Section V for emergency and first aid instructions.

RECOMMENDED FIELD PROCEDURES: The HD should be contained using


vermiculite, diatomaceous earth, clay or fine sand and neutralized as soon as possible
using copious amounts of 5.25% sodium hypochlorite solution. Scoop up all material
and clothing and place in a approved DOT container. Cover the contents of the
container with decontaminating solution as above. The exterior of the container will
be decontaminated and labelled according with EPA and DOT regulations. All leaking
containers will be over packed with vermiculite placed between the interior and
exterior containers. Decontaminate and label in accordance with EPA and DOT
regulations. Dispose of the material in accordance with Federal, state and local
regulations. Conduct general area monitoring with an approved monitor to confirm
that the atmospheric concentrations do not exceed the airborne exposure limits.

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SECTION VIII - SPECIAL PROTECTION INFORMATION


RESPIRATORY PROTECTION:

CONCENTRATION RESPIRATORY PROTECTIVE EQUIPMENT.


< 0.003 mg/m3 A full face piece, chemical canister, air purifying
protective mask will be on hand for escape.
(The M9-, M17-, or M40-series masks are
acceptable for this purpose. Other masks
certified as equivalent may be used)
> 0.003 mg/m3
A NIOSH/MSHA approved pressure demand full
face piece SCBA suitable for use in high agent
concentrations with protective ensemble.
(See DA PAM 385-61 for examples).

PROTECTIVE GLOVES: Butyl Rubber Gloves M3 and M4 Norton, Chemical


Protective Glove Set

EYE PROTECTION: As a minimum, chemical goggles will be worn. For splash


hazards use goggles and face shield.

OTHER PROTECTIVE EQUIPMENT: For laboratory operations, wear lab coats,


gloves and have mask readily accessible. In addition, daily clean smocks, foot covers,
and head covers will be required when handling contaminated lab animals.

MONITORING: Available monitoring equipment for agent HD is the M8/M9


detector paper, blue band tube, M256/M256A1 kits, bubbler, Depot Area Air
Monitoring System (DAMMS), Automated Continuous Air Monitoring System
(ACAMS),CAM-M1, Hydrogen Flame Photometric Emission Detector (HYFED), the
Miniature Chemical Agent Monitor (MINICAM), and Real Time Analytical Platform
(RTAP).

SECTION IX - SPECIAL PRECAUTIONS

PRECAUTIONS TO BE TAKEN IN HANDLING AND STORING: When handling


agents, the buddy system will be incorporated. No smoking, eating, or drinking in
areas containing agents is permitted. Containers should be periodically inspected for
leaks, (either visually or using a detector kit). Stringent control over all personnel
practices must be exercised. Decontaminating equipment will be conveniently placed.
Exits must be designed to permit rapid evacuation. Chemical showers, eyewash
stations, and personal cleanliness facilities must be provided.

24
DSTO-TR-0703

Appendix 2: CPNB Varian 3400/PFPD start up


procedure.

Pneumatics: Turn on the appropriate cylinders, on the east wall outside of Rm 417.
Hydrogen: 330 kPa
Nitrogen: 500 kPa
Air: 500 kPa
Air (for 8200): 440 kPa

3400 GC:
Action Result
Turn on GC The display reads:
(toggle switch at back of GC, top LHS) WARM START UP OCCURED
Turn on Auto Sampler Green light on front of autosampler
(switch at back of auto sampler, bottom LHS) will light up.
Press the activate key The display reads:
(in the Operations Section of the key pad) SELECT METHOD OR TABLE
Press the method 1 key The display reads:
(in the Method Section of the key pad) UNDER REMOTE CONTROL
Press the GC configure key The display reads:
(in the GC control section of the key pad) SET TIMES OR DATE? NO
Press the enter key The display reads:
(in the Entry Section of the key pad continue TURN HARDWARE ON-OFF? NO
until the Hardware On-Off line appears)
Press the yes key The display reads:
(in the Entry Section of the key pad) DETECTOR B ON? NO
Then press the enter key
Press the yes key The display reads:
(in the Entry Section of the key pad) DETECTOR OVEN ON? NO
Then press the enter key
Press the yes key The display reads:
(in the Entry Section of the key pad) INJECTOR OVEN ON? NO
Then press the enter key
Press the yes key The display reads:
(in the Entry Section of the key pad) AUXILIARY OVEN ON? YES
Then press the enter key
Press the no then enter keys The display reads:
(in the Entry Section of the key pad) GC CONFIG COMPLETE
Until the GC config complete line appears.

25
DSTO-TR-0703

PC/Star Workstation

Action Result
Switch ON Computer, wait until programs Screen displays:
load then minimize Program Manager Star Software
Click on System Control/Automation Screen displays:
then wait for software to load. System Control Configuration
Click on Instrument Screen displays:
then PFPD in drop down menu System Control PFPD
ACTIVATE METHOD: Screen displays:
Click of File, then Method File in the drop Activate a System Control Method
down menu, then activate. File dialogue box
Click on the method you want activated. Screen displays:
(C:\Star\PAL\Pal.mth or Pal-low.mth) System Control
then OK
Click on inject Screen displays:
Active Method Box
Once Active Method Box letters darken, Screen displays:
close window. System Control PFPD
ACTIVATE SAMPLE LIST: Screen displays:
Click of File, then Sample List File in the drop Activate a System Control Sample
down menu, then activate. List dialogue box
Click on the sample list you want activated. Screen displays:
(C:\Star\PAL\analysis.smp or Calib.smp) Sample List Window
then OK
Minimize sample list window Screen displays:
Note: samples may be added, deleted or System Control PFPD
modified through this window
ACTIVATE SEQUENCE FILE: Screen displays:
Click of File, then Sequence File in the drop Activate a System Control Sequence
down menu, then activate File dialogue box
Click on the sequence file you want activated. Screen displays:
(C:\Star\PAL\analysis.seq or Calib.seq) Active Sequence List Window
then OK
Minimize sequence window Screen displays:
Note: the sequence of events may be modified System Control PFPD
through this window
Click on Automation, then begin. Screen displays:
Instrument 1 Parameters
Click on OK Screen displays:
System Control
Click on OK Screen displays:
System Control PFPD

26
DSTO-TR-0703

Appendix 3: PFPD Mass Flow Controller Calibration

27
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Appendix 4: Star Chromatography Workstation Setup

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Appendix 5: Purity Determination of Sulfur Mustard


by GC-MS

322 Acquired on
100
05-Feb-1998
at 13:52:34
Scan EI+ TIC
5.53e6 Scan

0
4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0
Retention time (min)

29
Page classification: UNCLASSIFIED

DEFENCE SCIENCE AND TECHNOLOGY ORGANISATION


DOCUMENT CONTROL DATA 1. PRIVACY MARKING/CAVEAT (OF
DOCUMENT)

2. TITLE 3. SECURITY CLASSIFICATION (FOR UNCLASSIFIED REPORTS


THAT ARE LIMITED RELEASE USE (L) NEXT TO DOCUMENT
Gas Chromatographic Analysis of Sulfur Mustard in Diethyl CLASSIFICATION)
Phthalate
Document (U)
Title (U)
Abstract (U)

4. AUTHOR(S) 5. CORPORATE AUTHOR

Paul A Lancaster Aeronautical and Maritime Research Laboratory


PO Box 4331
Melbourne Vic 3001 Australia

6a. DSTO NUMBER 6b. AR NUMBER 6c. TYPE OF REPORT 7. DOCUMENT DATE
DSTO-TR-0703 AR-010-603 Technical Report August 1998

8. FILE NUMBER 9. TASK NUMBER 10. TASK SPONSOR 11. NO. OF PAGES 12. NO. OF
510/207/0896 ADF 95/065 SGADF 29 REFERENCES
2
13. DOWNGRADING/DELIMITING INSTRUCTIONS 14. RELEASE AUTHORITY

Director Aeronautical and Maritime Research Laboratory

15. SECONDARY RELEASE STATEMENT OF THIS DOCUMENT

Approved for public release

OVERSEAS ENQUIRIES OUTSIDE STATED LIMITATIONS SHOULD BE REFERRED THROUGH DOCUMENT EXCHANGE CENTRE, DIS NETWORK OFFICE,
DEPT OF DEFENCE, CAMPBELL PARK OFFICES, CANBERRA ACT 2600
16. DELIBERATE ANNOUNCEMENT

No Limitations

17. CASUAL ANNOUNCEMENT Yes


18. DEFTEST DESCRIPTORS

Gas Chromatography, Pulsed Flame Photometric Detector, Sulfur Mustard

19. ABSTRACT
A gas chromatographic method for the analysis of 2,2'-dichlorodiethyl sulfide (commonly known as
Sulfur Mustard or HD) that had been trapped in the solvent, diethyl phthalate (DEP) is described. The
method utilises the improved sensitivity and selectivity offered by the new Pulsed Flame Photometric
Detector to detect routinely samples containing 0.25 µg/mL of sulfur mustard in DEP. The method is
capable of fast and reliable analysis of samples containing 0.25-5.0 µg/mL of sulfur mustard in DEP.
Quality control measures determined that the method calibration was still within 5% control limits after
nearly 500 sample injections.

Page classification: UNCLASSIFIED