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Helsinki, June 2008
B. Spangenberg
A new method for the quantification of
Paraquat, Diquat, Difenzoquat,
Mepiquat and Chlormequat in water
by Thin-Layer Chromatography
Meltem Göcer, Kathrin Hoferer, Jürgen Zipfel and
Bernd Spangenberg* (Offenburg, Germany)
Structures of Quats, investigated
B. Spangenberg
-
2 Br
+ + +
N N
N
N
CH3
CH3
Diquat
CH3
+
N N
+
CH3 Difenzoquat
- -
Cl Cl
CH3
Paraquat
CH3 -
Cl
+ -
N Cl
+
Cl CH2 CH2 N CH3
CH3
CH3
Mepiquat Chlormequat
What are Quats ?
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Quats are used as quaternary ammonium
herbicides.
All of these are easily reduced to
the radical ion, which generates
superoxide radicals that reacts
with unsaturated membrane lipids.
Today Quats are among the most
commonly used herbicides.
Quats and the European Union
B. Spangenberg
The European Union allowed Paraquat in
2004. Sweden, supported by Denmark,
Austria, and Finland, brought the
European Union Commission to court.
On 11 July 2007 the court annulled the
directive authorising Paraquat as an
active plant-protection substance.
In the European Union, paraquat has been
forbidden since 10th of July 2007.
What are Quats ?
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All Quats are potential ground water
contaminants.
We actually don‘t know whether quats are a
ground water problem or not.
Challenges of quat analysis
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We focussed on four challenges in quat analysis:
1.) optimisation of the separation system
2.) optimisation of the detection system
3.) improvements in the detection limits
4.) optimisation of sample pre-treatments
Challenges of quat analysis
B. Spangenberg
We focussed on four challenges in quat analysis:
1.) optimisation of the separation system
2.) optimisation of the detection system
3.) improvements in the detection limits
4.) optimisation of sample pre-treatments
Influence of salt content on the mobile phase
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Stationary phase:
LiChrospher®, Merck
Mobile phase:
1-propanol, methanol
and (A – F), (1+1+3, V/V)
5
A: water No. name
4
B: 0.5 m NaCl 1: paraquat
3
C: 1.0 m NaCl 2: diquat
1+2 D: 1.5 m NaCl 3: mepiquat
E: 2.0 m NaCl 4: chlormequat
A B C D E F F: 2.5 m NaCl 5: difenzoquat
A
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Methanol, 2-propanol, 2.5 n NaCl/H2O (1+1+3) Methanol, 1-propanol, 2.5 n NaCl/H2O (1+1+3)
9 2,5
8
7 2
6
5 1,5
4
1
3
2
0,5
1
0 0
0 10 20 30 40 50 60 0 10 20 30 40 50 60
cm cm
chlormequat+difenzoquat chlormequat difenzoquat
Challenges of quat analysis
B. Spangenberg
We focussed on four challenges in quat analysis:
1.) optimisation of the separation system
2.) optimisation of the detection system
3.) improvements in the detection limits
4.) optimisation of sample pre-treatments
Dragendorff-staining of quats (detection limits)
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Dragendorff-staining
The amount of 0.85 g basic
Difenzoquat (80 ng) bismuth nitrate is dissolved
in 10 mL acetic acid and 40
Mepiquat mL water. For solution b:
(800 ng) 16g potassium iodide is
dissolved in 40 mL water.
The final reagent is mixed
from 2 mL solution a and 2
mL b. Then 8 mL acetic acid
Diquat (30 ng) are added and this mixture
is topped up with water to 50
Paraquat (20 ng) mL. Solution a and b remain
paraquat stable for several weeks.
Chlormequat remains invisible!
Formation of tetraphenyl-diboroxyde
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+
Na
H+/H2O
- B
B
? B
O
sodium tetraphenyl-borone tetraphenyl-diboroxyde
To 50 mg sodium tetraphenyl-borone (Na[B(C6H5)4] in 50 mL
water 50 µl HCl (32 %) were added.
Formation of tetraphenyl-diboroxyde: R. Neu, Chem. Ber. 87 (1954), 802 - 805
Formation of tetraphenyl-diboroxyde
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To 50 mg sodium
tetraphenyl-
borone
(Na[B(C6H5)4] in
50 mL water 50
µl HCl (32 %)
were added.
After 24 hours
the solution
turns turbid.
after 24 h after 8 h
Formation of tetraphenyl-diboroxyde: R. Neu, Chem. Ber. 87 (1954), 802 - 805
Staining reaction with primulae flos
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Na-tetraphenylborone,
dissolved in 50 mL
water + 50 µl HCl
(32%).
Na+ tetraphenyl- tetraphenyl-
borone +HCl diboroxyde
This reagent stains
more specifically
than NEU-reagent or
tetraphenyl-
diboroxyde.
R. Neu, Z. anal. Chem. 143 (1954), 30 - 38
A new sodium tetraphenylborone staining
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Detection wavelengths : 470 – 530 nm To 50 mg sodium
tetraphenyl-borone
(Na[B(C6H5)4] in 50 mL
difenzoquat water 50 µl HCl (32 %)
were added.
chlormequat The wet plate is
illuminated for 5 minutes
by intense light of 254 nm.
mepiquat Spots of mepiquat,
chlormequat and
diquat difenzoquat are converted
into fluorescing zones.
para- Paraquat and diquat spots
quat were illuminated for 10
minutes with UV-light of
365 nm.
Idea from: R. Neu, Z. anal. Chem. 143 (1954), 30 - 38
Challenges of quat analysis
B. Spangenberg
We focussed on four challenges in quat analysis:
1.) optimisation of the separation system
2.) optimisation of the detection system
3.) improvements in the detection limits
4.) optimisation of sample pre-treatments
High intensity LED for TLC-fluorescence measurements
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Light intensity: 100 mW !
The diode shows absolutely constant
light intensity!
Fluorescencence 3D plot of Quats
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Difenzoquat
Paraquat
Mepiquat Chlormequat Diquat
Fluorescencence contour-plot of Quats Front
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Difenzoquat
Diquat Paraquat
shows a
green-yellow,
Chlormequat
diquat a green
and all other
quats a blue
fluorescence.
Paraquat Mepiquat
Densitogram of Quats from a real water sample
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2,5
Application Stationary phase:
2 Silica gel „LiChrospher®“,
Front Merck company
1,5
Mobile phase:
1
Methanol, 1-propanol,
2.5 n NaCl/H2O (1+1+3)
0,5
40 min developing time
0
0 10 20 30 40 50 60
cm
Paraquat Mepiquat Difenzoquat
Diquat Chlormequat
Detection range of paraquat
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50
45 detecting range 1 – 200 ng
40
35
30
(R-1)
25 4
20
3,5 detecting range 1 – 20 ng
15
3
10
5 2,5
(R-1)
0 2
0 50 100 150 200 250
1,5
ng paraquat
1
0,5
0
Plate was dipped in a solution of 0 5 10 15 20 25
ethylene glycol/methanol 1+1, (V/V). ng paraquat
Linear regression and estimation of detection and quantification limits, according to W. Funk et. al.
„Statistische Methoden in der Wasseranalytik“, VCH Weinheim 1985
Detection range of diquat and mepiquat
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40
35
detecting range 0,5 – 40 ng
30
25
(R-1)
20
15 50
10
45 detecting range 30 – 500 ng
5 diquat 40
35
0 30
(R-1)
0 10 20 30 40 50
25
ng diquat 20
15
10
5 mepiquat
0
0 100 200 300 400 500
ng mepiquat
Detection range of chlormequat and difenzoquat
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25
detecting range 10 – 370 ng
20
15
(R-1)
10 100
90 detecting range 30 – 530 ng
5 80
chlormequat 70
0 60
(R-1)
0 100 200 300 400 50
ng chlormequat 40
30
20
10
difenzoquat
0
0 50 100 150 200 250 300 350 400 450 500 550
ng difenzoquat
Challenges of quat analysis
B. Spangenberg
We focussed on four challenges in quat analysis:
1.) optimisation of the separation system
2.) optimisation of the detection system
3.) improvements in the detection limits
4.) optimisation of sample pre-treatments
Optimisation of sample pretreatments
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Water samples are enriched by use of MAC-3 c,
kationic resin, standard grade (Dow Chemical
Rheinmünster, Germany). The resin is packaged in
a plastic cartridge (3 g), the volume 1000 mL
sample is passed through the resin cartridge and
eluated with 50 mL 0.1 m HCl.
The sample evaporation is done by use of a gentle
stream of air over the eluate surface. The dry
sample is topped up to 30 mL with methanol,
filtered, evaporated to dryness and topped up with
500 µl methanol.
The amount of 20 µl sample is applied on plate.
Detection limits and quantification limits of quats
B. Spangenberg
name detection limit quantification limit (20 µL)*
========================================================
Paraquat 6.25 ng 9.0 ng 220 ng/L
Diquat 2.25 ng 3.25 ng 82 ng/L
Mepiquat 35 ng 50 ng 1.25 µg/L
Chlormequat 25 ng 30 ng 0.78 µg/L
Difenzoquat 90 ng 105 ng 2.60 µg/L
*: for 1 L water, extracted in 500 µl methanol and 20 µl applied on plate
Detection levels in drinking water (according to US EPA method 549.2):
Paraquat 680 ng/L and diquat 720 ng/L
Estimation of detection and quantification limits, according to W. Funk et. al.
„Statistische Methoden in der Wasseranalytik“, VCH Weinheim 1985
Recovery rate
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Name RF-value recovery rate rel.sdv.
Paraquat 12.0 98.9 % (20.2 µg/L) 40.6 %
Diquat 16.3 26.7 % (4.9 µg/L) 28.0%
Mepiquat 28.4 107.4 % (49 µg/L) 12.9%
Chlormequat 43.9 63.8 % (36 µg/L) 18.8%
Difenzoquat 67.5 115.6 % (53 µg/L) 48.5%
Conclusion
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- all five quats are quantitatively detectable
- the detection limits are quite good
- the sample preparation step (and the recovery rate)
is insufficient
B. Spangenberg
Thank you very much
for your attention
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