THIN LAYER CHROMATOGRAPHY OF CHEMICAL WARFARE AGENTS
Javier C. Quagliano1, Zygfryd Witkiewicz2 and Stanisław Popiel2
1. Dpt. of Applied Chemistry, Argentine R&D Institute for the Defense (CITEFA), 2. Institute
of Chemistry, Military Technical Academy, Warsaw, Poland.
Keywords: Thin layer chromatography, chemical warfare agents, organophosphorous
Abstract
In this contribution advances in thin layer chromatography (TLC) of chemical warfare agents
(CWA) are reviewed. Procedures for separation and identification of CWA are described,
alone and in the presence of related compounds, such as organophosphorous pesticides for the
case of nerve agents. Analysis of blister agents (vesicants) and their differentiation, irritants
(CS, CN and CA) and mixtures of irritants and nerve agents are referred. Solvents for running
the TLC plates, reagents and conditions for development are thoroughly described. Utilization
of Two-dimensional overpressured TLC (OPLC) for separation and identification of nerve
agents in the presence of organophosphorous pesticides proved to be an adequate technique
for detecting very low concentrations of these compounds.
Introduction
Chemical warfare agents (CWA), commonly used during the First World War, were also
prepared for possible use during the WWII. However, they were not used in battlefield during
that war. Only hydrogen cyanide was used for killing prisoners held in German death-camps,
for example in Auschwitz. Despite existing proofs that a war can be fought without use of
CWA, the agents were industrially produced after the WWII. Those were used on a local
scale, and still remain in store of some armies. Therefore, usage of CWA cannot be excluded
in local armed conflicts. Cases of CWA usage by terrorists have also been noted [1]
.
International community revoked development, production, storage and usage of
CWA with a Chemical Weapons Convention, which entered into force in 1997. One of the
basic tasks foreseen by the Convention is destruction of CWA stocks owned by Member
States of the Convention. Inspectors of the Organization for the Prohibition of Chemical
Weapons, monitoring observance of provisions of the Convention, perform analyses of
chemical substances, which could potentially be CWA. Analyses of various substances are
also performed in those places, where usage of chemical warfare is suspected. One of the
analytical methods allowing detection and determination of precursors for production of
CWA, CWA themselves and their decomposition products is thin layer chromatography
(TLC).
Currently, gas chromatography (GC), included gas chromatography mass spectrometry
(GC-MS), is the main method used for CWA analysis [2,3]. High performance liquid
chromatography with mass spectrometry (HPLC-MS) is a method more and more frequently
used to that purpose. Those analytical methods are very efficient, but expensive and require
some complex apparatuses.
Thin layer chromatography (TLC) is a relatively simple and cheap analytical method,
although increasingly elaborate devices are used in it, and it is a fully instrumental method.
High selectivity, good detectability of analytes and reliability of the analysis, even if a
relatively simple equipment is used, makes the method applicable not only for stationary
laboratories, but also for mobile field laboratories.
General characteristic of TLC as a CWA analytic method
A TLC analysis can be performed right at a site of occurrence of suspicious chemicals,
checking – for example – 34-40 samples in 4-5 hours. The analysis provides information on
presence or absence of interesting chemicals, and dubious samples can be further analysed
using other chromatographic techniques, such as GC- MS. Analysis using TLC is particularly
recommended if numerous samples of unknown origin have to be analysed. Therefore, in
some armies, including Polish army, TLC apparatuses and procedures have been and still are
used for CWA analysis in field laboratories.
TLC analyses samples having a form of a solution. If analysed material is located on a
chemical reactor wall, surface of contaminated equipment or remains of a missile, such a
surface is wiped with cloth or polyester and then a chemical substance is extracted with
proper solvent, e.g. dichloromethane, hexane or acetone. Also samples of soil and water are
extracted with solvents. Those extracts can be concentrated, if necessary.
Obtained extracts are applied onto a chromatographic plate covered by a thin layer of
adsorbent (volume 1 to 5 µl). Silica gel is most frequently used as an adsorbent, and
aluminium oxide is used less frequently. Components of a sample mixture are separated by
development of a chromatogram using a single or mixed mobile phase characterised by
appropriate polarity and elution force. After development of a chromatogram, colourless
components are transformed into their colour products. To achieve that, the chromatographic
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plate is sprayed with a solution of an appropriately selected reactant. Positive colour reaction
can occur immediately, after some time (depending on quantity of analyte), or only after the
plate is heated up. Detectability limit of TLC is usually between 10 -6 to 10-9 g in a spot, and
the value for HPTLC is between 10-9 to 10-12 g.
Identification of a sample component on a plate is realised by comparison of the
obtained colour with a specific standard. To increase reliability of identification, several
reactants can be used, providing products of various colours characteristic for a given
chemical warfare agent.
Besides a colour of analyte, identification is also based on delay factor of a given spot
comparing to the front of the mobile phase (RF). Value of the factor is then compared to the
corresponding value of this factor for a standard.
In case when a toxic substance occurs alone, without any admixtures, it can be
detected on a chromatographic plate without development of a chromatogram. A drop
analysis is performed, using one or more reactants providing colour products with the
detected (identified) substance.
Besides visualisation of components on a chromatogram realised by their transition to
colour chemicals, also other visualisation methods are used, for example: irradiation with UV
light. After irradiation of a developed chromatogram, spots invisible in visible light can
become fluorescent, or can extinguish background fluorescence of a plate surface.
Components of a single sample can be detected and identified on a single plate using various
methods.
TLC is used for qualitative, semi-quantitative and quantitative analyses. Precise,
accurate and repeatable results of quantitative analysis are achieved using densitometers. This
way – spot chromatograms can be transformed into peak chromatograms and absorption
spectra of analytes can be registered. Those spectra are used for identification of analysed
substances.
Detailed description of TLC as an analytical method is presented, among others, in a
monograph edited by Nyiredy [5].
Examples of analysis of individual groups of CWA using TLC are provided below.
Analysis of organophosphorus compounds
Combination of mixture components separation using HPTLC with enzymatic
visualisation is a very good method of analysis of organic phosphorus compounds. The
method is characterised by low detectability thresholds. Organic phosphorus compounds are
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inhibitors of enzymes – cholinesterase or acetylcholinesterase. [6,7]. Therefore, their
detection on a plate is limited to finding those places in a chromatogram, where an enzyme
was applied. Analysis of Sarin, Soman, Tabun and VN1 is performed, for example, using a
plate covered by a layer of silica gel and diatomaceous earth and a mobile phase containing
hexane, piridine and dioxane (7:2:1). After the plate is dried, it is sprayed with aqueous
solution of an enzyme (cholinesterase), and after next 5 minutes, with a mixture of -naphthol
acetate and diazo-o-dianizidine in aqueous-alcohol solution. White spots contrasting on a
intensive violet gel appear in those places, where organic phosphorus toxic compounds are
located. This visualisation method allows detection of 10 ng of an organic phosphorus
compound per spot. In the described conditions, VN1 appeared as two spots of isomers –
thiolic and thionic.
Enzymatic reaction can be utilised to analysis of not only organic phosphorus
chemical agents, but also organic phosphorus pesticides and carbamates, as those compounds
also inhibit cholinesterase, and can occur together with CWA in field conditions. Therefore,
methods have been developed to differentiate choline esterase-blocking pesticides and organic
phosphorus CWA [8]. Ten insecticides and Soman and VX were separated on plate with silica
gel. A mixture of dichloroethane and ethyl acetate (9:1) was used as a mobile phase. Analysed
chemicals were identified with selective reactions. Total time of the analysis did not exceed
30 minutes.
Enzymatic visualisation of organic phosphorus warfare agents (Sarin. Soman, Tabun
and VX) and organic phosphorus pesticides (Diazinon, Dichlorvos, Chlorfenvinphos,
Fenitrothion and Phosalone) uses 4-methylumbelliferone esters as reagents. The performance
of enzymatic reaction on a chromatographic plate was estimated by the fluorescence
measurement of the reaction product, which was 4-methylubelliferomne. The effects of
reaction were compared with the effects of reactions when indoxyl acetate was used. Using
the procedure it was possible to determine sarin, soman and tabun at the level of dozen
picograms per spot and VX as well as pesticides from the individual nanograms to their
dozens per spot [9].
Two-dimensional overpressured TLC (OPLC) has been used for the separation and
quantitative densitometric analysis of organophosphorus warfare agents in the presence of
twenty two pesticides [10]. The CWA were: tabun, sarin, soman, DFP, VX and the pesticides
belonged to the groups of organochlorine (5), organophosphorus (13), carbamate (2) and
carbamide (2) compounds. The optimum composition of the mixed eluents was established
using the PRISMA model. The chromatograms were developed to a distance 6 cm in the first
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direction with diisopropyl ether – benzene – tetrahydrofuran – n-hexane (10 + 7 + 5 + 11, v/v)
and to a distance 6 cm in the second direction with tetrahydrofuran – n-hexane (2 + 3, v/v).
Three spray reagents were used for visualization of analysed compounds. Reagent A was a
solution of cholinoesteraze in borate buffer, reagent B was a solution of butyrythiocholine
iodide and Michler’s hydrol and reagent C was a saturated solution of cobaltic chloride in
acetone. After evaporation of the mobile phase, the plate was sprayed with reagent A, placed
in an oven at 37 oC for to 15 min and then sprayed successively with reagent B and C. The
presence of analytes was revealed as blue spots on the white background. The detectability
was 1.3 pg for tabun and 48 ng for VX and it was possible to determine the organophosphorus
warfare agents quantitatively in the range of 15 pg to 100 ng [JPC 4].
It was found out that the presence of metal ions in the enzymatic visual reagent leads
to decrease of the detection limit of organophosphorus compounds to the level 10-12 g per
spot. The best results were obtained for Co+2 and slightly worse for Mg+2 ions [11,12].
DFP can be determined using highly sensitive non-enzymatic method, after its
visualisation in reaction with 2,4-dinitrophenol (DNPS) and 2,4,6-trinitrophenol (TNPS) [13].
Spots – brown (DNPS) or orange (TNPS) – disappeared in several minutes. Re-staining could
be achieved spraying a plate with NaOH solution. Determinability of DFP was between 1 and
2 nmol per spot.
Analysis of blister agents (vesicants)
This group of CA includes mustards and arsenic compounds, especially lewisites.
Various reagents were used for visualisation of sulphur and nitrogen mustards. A group
reagent for sulphur and nitrogen mustards can be 4,4’-nitrobenzylpiridine, giving blue
coloured products with mustards. Sulphur mustards can be differentatied from nitrogen ones
by spraying plates with Cu-3,3’-dimethoxybenzidine. Usage of those reagents allows
detection of mustards at a level of micrograms per spot [14]. Also a biochemical method was
used for development of thin layer chromatograms of compounds having cytotoxic activity.
The method allowed determination of sulphur mustard at the level of single μg.
Munawalli and Pannel [15] analysed sulphur mustard and its metabolites in biological
fluids using TLC. Very good separation of those substances were achieved using twocomponent mixture of chloroform and methanol (10:1) and chloroform and acetylnitrile (5:1)
for development of chromatograms. Visualisation of those chromatograms was achieved
using 1 % KMnO4 solution in 6 % Na2CO3 and 7 % 4,4’-p-nitrobenzylpiridine solution in
acetone. Heating a plate for 15 minutes at 70oC and exposure to ammonia vapours caused
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formation of blue stains in those places, where sulphur mustard was present. This method
allowed detection of less than 0.05 g of yperite per spot.
Significant amount of CWA, mostly sulphur mustard, was sunk in the Baltic Sea after
the WWII [16,17]. An analysis of a yperite block fished in 1997 from the Baltic Sea was
performed using HPTLC and OPLC [18]. By use of two-dimensional elution it was possible
to detect from 13 to 22 different compounds in samples from the block. The detected
compounds were identified by gas chromatography with mass spectrometry. Sulphur mustard
was found to be the major component of the yperite block. It was determined at a limit of
detection of 20 ng per spot. The maximum quantity of sulphur mustard in the analyzed
samples did not exceed 26 %. Plates with silica gel were used for analysis. There were the
following mobile phases: for development in one direction - toluene – dichloromethane – npropanol – n-hexane (25 + 25 +1 + 50, v/v); and in second direction - diisopropyl ether –
chloroform – n-hexane (1 + 1 + 3, v/v). The chromatograms were visualized by spring with
reagent containing 4,4’-bis(diethylamino)benzophenone and mercury(II)chloride in ethanol.
Better separation results for components of mixture and in shorter time were achieved using
OPLC, comparing to HPTLC. It is interesting to note, that after staying for approx. 50 years
in marine water, the block contained significant amount of yperite.
The group of necrosis-inducing CWA includes also primary, secondary and tertiary
arsines. Those chemical compounds were also analysed using TLC. Separation of mixtures of
those arsines is not difficult and they can be visualised using Michler’s thioketone, iodine,
dithizone and a meta cresol purple dye. Using the latter reagent it is possible to detect lewisite
at the level of 0.05 μg per spot [19].
Analysis of irritants
Chemicals belonging to that group differ with polarity, which requires an appropriate
mobile phase at one hand, and favours their separation on the other. Good separation of a
mixture of CS, CA and CN can be achieved using 5% chloroform in benzene as a mobile
phase. Developing a chromatogram with quinone solution a yellow CA and a blue CS spot is
achieved. Following additional spraying with 5% NaOH solution a brown CN spot appears.
DM and diphenylcyjanoarsine can be separated using 20% acetone in chloroform and
visualised with solution of 3,3’-dimethoxybenzidine, copper acetate and 50 % H2SO4,
solution, receiving an orange spot.
CA, CS and CN present in a single sample become separated when chromatogram is
developed using a mixture of toluene and dichloroethane (1:1). After a plate is sprayed with
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thiourea and 3,3’-dimethoxybenzidine solution, yellow CA, beige CS and brown-violet CN
spots are obtained. Detectability of those substances is approx. 1 g per spot.
A mixture of CR, CS and CN can be separated using chloroform as a mobile phase.
Dragendorff reagent or KMnO4 solution can be used for development. Separation takes place
acc. to the following increasing RF values: CR  CN  CS.
Analysis of mixtures containing CWA belonging to particular groups
In case of analysis of a blend of 12 organic phosphorus compounds, necrosis-inducing
coampounds and irritants using enzymatic reaction, besides spots corresponding to organic
phosphorus compounds also spots corresponding to lacrimators - CS and CN - appear. Spots
of those agents overlap with spots of Sarin and Soman, and therefore analysis of such a blend
should be carried out in two chromatographic systems. In the first one a mixture of n-hexane,
dioxane and pyridine (&:1:2) can be used as a mobile phase. Development of a chromatogram
using enzymatic reaction leads to appearance of GB, GA, GD, VN1, CS and CN spots, and
spraying the chromatogram with Tollens reagent leads to appearance of mustard, CA, CS, CN
and DM spots. In a second system, using dichloroethane and ethyl acetate (7:3) as a mobile
phase, separation of CS, CN, DM and GB as well as GA is achieved. In this system, RF values
for lacrimators and organic phosphorus toxic agents were clearly different. Visualisation of a
chromatogram can be realised using enzymatic reaction. Preliminary spraying a plate with I2
solution in chloroform increases sharpness of silhouettes of spots and makes visualisation of
adamsite possible.
Analysis of sulphur mustard can be realised when the compound is present in a blend
containing organic phosphorus and/or organic chlorine insecticides [20]. Identification of
sulphur mustard was possible was possible due to application of a mobile phase ensuring its
good separation from pesticides. RF value of yperite was high and for the other components of
the blend it was low. Mustard spot was visualised using a selective reagent, for example: a
iodineplatinate ion [PtJ6]-2. This allowed detection of yperite at a sub-microgram level.
Conclusions
Presented data concerning CWA analysis using TLC method indicate that there are
numerous procedures allowing separation, identification and determination of CWA. This is
possible even in those situations, when CWA are present in composed mixtures, containing
other substances, that can be present in environmental samples. Analyses can be completed in
mobile laboratories, many armies are equipped with.
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Although no descriptions of new methods of CWA analysis are published recently, but
analysis methods for similar chemical compounds using TLC are being developed. A
procedure intended for analysis of organic phosphorus pesticides described by Hamada and
Wintersteiger could be used for analysis of organic phosphorus toxic agents [21].
For analysis of organic phosphorus compounds In human serum after acute poisoning
the procedure proposed by Fugatami at al. [22] may be used. They applied HPTLC for
detection twenty five commonly used organic phosphorus insecticides in human serum. These
organophosphates were separated on plates with three different separating systems within 6 –
18 min and detected by means of ultraviolet radiation and coloring reactions.
Not only common TLC, but also some more advanced techniques can be used for
CWA analysis. A combination of TLC with flame ionization detection was suggested for
analysis of phosphorus and sulphur compounds[23].
Besides a typical analysis of CWA using TLC, chromatographic plates used for
analysis can be also used for preliminary, rapid detection of CWA. Chromatograms are not
developed in that case, but tested samples are applied in form of a solution over a layer of
adsorbent and then wetted with solutions of appropriate reagents. Characteristic green spots
appear in case of presence of a toxic agent [24].
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