Capillary electrophoresis technique for rapid separation and detection of organophosphate nerve agents

1. Capillary Electrophoresis technique for rapid separation and
detection of Organophosphate Nerve Agents.
Anna Hogan
Sensing and Separation Group,
Department of Chemistry and
Life Science Interface Group,
Tyndall National Institute
University College Cork
Ireland
15/04/16
CASI

2. Classification in terms of physiological effects produced on humans by the Chemical
Warfare (CW) agents used in warfare :
•Nerve agents
Organophosphate compounds
•Vesicants (blistering agents)
mustards and arsenicals
•Bloods agents (cyanogenic agents)
hydrogen cyanide and cyanogen chloride
Chemical Warfare

3. Chemical Warfare
•Choking agents (pulmonary agents)
chlorine, phosgene, diphosgene, nitric oxide,
and perfluoroisobutylene
•Riot-control agents (tear gases)
2-chloroacetophenone (CN),
and 2-chlorobenzilidenemalononitrile (CS)
•Psychomimetic agents
lyserigic acid diethylamide (LSD)
and 3-quinuclidinyl benzilate (BZ)
•Toxins
botulinum neurotoxin (agent X)

4. Toxic effects
Types of toxic effects observed with pesticides and nerve gases:
•Inhibition of cholinesterases:
The accumulation of acetycholine leads to:
 muscarinic
 nicotinic
 central nervous system effects
•Delayed neuropathy
Caused by certain OP compounds, such as tri-orthocresyl phosphate. The OP
compounds interact with a neuropathy target esterase disturbing metabolism
in the neurone.
 degradation of peripheral nerves in the distal parts of lower limbs which may
spread to the upper limbs. (“ Jake paralysis”)
•Treatment
 pralidoxime
 atropine

5. Nerve agents
Paraoxon-methyl (MPX)
Paraoxon-ethyl (EPX)
Parathion-methyl (MPT)
Parathion-ethyl (EPT)
Fenitrothion (FT)
Organophosphorus insecticides are the most widely used and the most frequently
involved in fatal human poisonings.

6. CE -UV
Capillary electrophoresis
High
Voltage
Detector
Electroosmotic flow
Cathode (-)Anode (+)
Buffer Buffer
+
N
N
_
-
+
Computer
Capillary
Figure 1: Schematic representation of capillary electrophoresis system with optical detection. The
circled +’s ,N’s, and –‘s represent cationic, neutral, and anionic solutes, respectively.
Figure 2: Schematic of separation principle in micellar electrokinetic capillary chromatography MEKC*.
*Handbook of capillary and microchip electrophoresis and associated microtechniques. Lander JP.
Boca Raton:CRC Press 2008.

7. CE-UV
Figure 3: Variation of different buffer concentrations Capillary: 8.5 cm
effective length x 50 μm i.d.; peak: 1 – MPX, 2 – EPX, 3 – MPT, 4 - FT
and 5 – EPT; buffer: 35 mM sodium acetate (pH 5), 10 mM SDS;
injection: 10 mbar for 4 s; Detection: 263 nm; temperature: 15 .℃
Overlays are offset, where the scale of y= 5 mAU units.
Figure 4: Effect of applied voltage on migration time. Capillary with
effective length of 8.5 cm x 50 μm i.d.; peak: 1 – MPX, 2 – EPX, 3 – MPT,
4 - FT and 5 – EPT (from left to right); buffer: 35 mM sodium acetate
(pH 5), 10 mM SDS; injection: 10 mbar for 4 s; Detection: 263 nm;
temperature: 15 . Overlays are offset, where the scale of y= 5 mAU℃
units.

8. CE-UV
Figure 5: Electropherogram of OPs ; capillary 8.5 cm x 50 μm i.d.;
peaks: 1 – MPX migrating at 0.80 min, 2 – EPX migrating at 1.34 min
, 3 – MPT migrating at 1.54 min, 4 – FT migrating at 2.06 min and 5
– EPT migrating at 2.39 min ; buffer: 35 mM sodium acetate (pH 5),
10 mM SDS; injection: 10 mBar for 4 s; voltage 20kV; Detection:
263 nm; temperature: 15 .℃
1
2
3
4
5
Organo-
phosphate
Nerve
Agents
t
(min)
b a r Range
(mM)
LOD
(mM)
Paraoxon-
methyl
Paraoxon-
ethyl
Parathion-
methyl
Fenitrothion
Parathion-
ethyl (EPT)
0.8
1.34
1.54
2.06
2.39
-0.0495
-0.3186
-0.3441
-0.3167
-0.318
63.757
93.971
72.365
69.1
72.318
0.9959
0.9710
0.9871
0.9701
0.9971
0.02 – 0.1
0.02 – 0.1
0.02 – 0.1
0.02 – 0.1
0.02 – 0.1
0.01
0.01
0.04
0.04
0.04
Calibration plots are expressed as linear regression equation (y= a + bx), where,
y is peak are and x is the analyte concentration mM.
Table 1: Parameters of CE method: migration time (t), parameters of analytical
curves (b-slope and a-intercept), correlation coefficients (r), the working range
(mM), precision (RSD), Limits Of Detection (LOD).

9. MCE - UV
Microchip Capillary electrophoresis
Figure 7: The real time progress of the separation length-based
electropherograms of peaks on a 45 sec timescale: 1 – MPT, 2 – EPX, and 3 – MPX
(from right to left); buffer: 45 mM acetate buffer (pH 5), 10 mM SDS; Sample
introduction: sample inlet (SI): 1 kV; sample outlet (SO): 0 kV, buffer inlet (BI):
0.34 kV, buffer outlet (BO):0.62 kV for 5 s; Separation settings: (SI): 0 kV, (SO): 0
kV, BI: 1 kV, BO:0 kV for 45 s; Detection: 214 nm.0 12.5 25
Separation length/mm
35sec
25sec
15sec
5sec
Figure 6: Photographs of electrophoresis microchip (Shimadzu Instruments, Kyoto,
Japan) used in experiment dimension 35 and 12.5 given in mm. Four platinum
electrodes on the chip to apply voltages between the sample introduction and
separation reservoirs, the chip encased in a propylene frame.
Sample injection port
Sample introduction channel
UV monochromatic light
Separation channel
Diode array with 1024 elements
1
2
3

10. MCE-C4
D
Figure 8: Photograph of portable microchip electrophoresis device.

11. MCE-C4
D
Future Work
•Microchip materials
PDMS
PMMA
PEEK
•Manufacture of electrodes
copper
aluminium
carbon
•Dimensions of electrodes
length
width
gap
•Further miniaturization of detector and control instrumentation

12. MCE-C4
D
Advantages of home-made box for microchip capillary electrophoresis with
contactless conductivity detection compare to conventional CE:
 Shrink in size and in price
 For on-site use
 Faster
 Simpler
 Flexibility of chip holder
 Disposability of plastic chips
 Effective isolation of sensing electrodes from high separation voltages
 Elimination of surface fouling

13. Detection of Organophosphate Nerve Agents

14. Acknowledgements
Project team Eric
Patricia Anna Walter
(lab-on-chip) (separation) (sensor)
Xi Yineng
This research is supported by European Commission
Contact: anna.hogan@tyndall.ie

15. Cork, Ireland, 8 - 9 March 2017

16. Thank you for your attention