Separation of the phenoxy acid herbicides and their enantiomers by capillary zone electrophoresis in presence of highly sulphated cyclodextrins

Article abstract of DOI:10.1002/jccs.200900167

The study of the chiral compounds and their fate in the environment is receiving an increasing attention - enantiomeric ratios are being measured and enantioselective degradation processes are being reported. It is particularly important with the toxic compounds like the pesticides, which are being freely used in the environment to control the harmful pests. Capillary zone electrophoresis was used for the chiral and mutual separation of four phenoxy acid herbicides using highly sulphated cyclodextrins (HSCD) in the buffer. The CE runs were performed with reverse polarity (anode in the outlet vial) using the acidic ammonium formate buffer (20 mmol, pH 3). Under these conditions of suppressed the electroendoosmotic flow (EOF), the analytes are mobilized to the anode by entering into host guest relation with the migrating negatively charged sulphated cyclodextrin. The phenoxy acid herbicides selected for the purpose were fenoprop, dicloprop, mecoprop and 2,4-DB. The α-HSCD and β-HSCD have been tested as resolving agents in the CE for the separation of the enantiomers of the herbicides. Though the chiral separation of the dicloprop and mecoprop were achieved with α-HSCD but it was not able to resolve fenoprop. With β-HSCD the required base line separation was achieved. Potential difference selected was 10 kV. The limit of detection (S/N=3) achieved in present case is 0.15 ppm for fenoprop, 0.14 ppm for dicloprop and mecoprop and 0.11 ppm for 2,4-DB.

Full text of DOI:10.1002/jccs.200900167

Journal of the Chinese Chemical Society, 2009, 56, 1163-1167
1163
Separation of the Phenoxy Acid Herbicides and Their Enantiomers by
Capillary Zone Electrophoresis in Presence of Highly Sulphated
Cyclodextrins
Ashok Kumar Malik,a* Jatinder Singh Aulakh,a Agnes Feketeb and Philippe Schmitt-Kopplinb
aDepartment of Chemistry, Punjabi University Patiala, Punjab, India
bGSF - National Research Center for Environment and Health Institute for Ecological Chemistry
Ingoldstädter Landstraße 1 D-85764 Neuherberg, Germany
The study of the chiral compounds and their fate in the environment is receiving an increasing atten-
tion — enantiomeric ratios are being measured and enantioselective degradation processes are being re-
ported. It is particularly important with the toxic compounds like the pesticides, which are being freely
used in the environment to control the harmful pests. Capillary zone electrophoresis was used for the
chiral and mutual separation of four phenoxy acid herbicides using highly sulphated cyclodextrins
(HSCD) in the buffer. The CE runs were performed with reverse polarity (anode in the outlet vial) using
the acidic ammonium formate buffer (20 mmol, pH 3). Under these conditions of suppressed the electro-
endoosmotic flow (EOF), the analytes are mobilized to the anode by entering into host guest relation with
the migrating negatively charged sulphated cyclodextrin. The phenoxy acid herbicides selected for the
purpose were fenoprop, dicloprop, mecoprop and 2,4-DB. The a-HSCD and b-HSCD have been tested as
resolving agents in the CE for the separation of the enantiomers of the herbicides. Though the chiral sepa-
ration of the dicloprop and mecoprop were achieved with a-HSCD but it was not able to resolve fenoprop.
With b-HSCD the required base line separation was achieved. Potential difference selected was 10 kV.
The limit of detection (S/N = 3) achieved in present case is 0.15 ppm for fenoprop, 0.14 ppm for dicloprop
and mecoprop and 0.11 ppm for 2,4-DB.
Keywords: Phenoxy acid herbicides; Enantiomer separation; Capillary electrophoresis;
Cyclodextrins.
INTRODUCTION
Capillary electrophoresis (CE) is widely used in vari-
recognition are cyclodextrins, including underivatised neu-
tral CDs, and derivatised charged CDs, as chiral additives
to the separation buffers.7
ous fields as one of the most efficient separation technique.
Number of publications on enantioseparations1,2 by CE has
appeared in recent years. UV absorbance is the most com-
monly used detection system in most commercially avail-
able CE instruments. Chiral separation is one of the objec-
tives of the CE as well as of the chromatographic methods.
Chiral solid phases3,4 for GC and HPLC columns and re-
agents allow the chromatographic separation of optical iso-
mers. This has given way to the fertile field of investiga-
tion, especially for the pharmaceutical products and amino
acids. The techniques for chiral separation by the CE, usu-
ally involves the addition of the chiral reagent to the sepa-
ration buffer5 and has been widely applied in the pharma-
ceutical and biomedical fields during the past few years.6
One of the most popular reagents for achieving the chiral
The ability of the native and derivatised cyclodex-
trins to form host guest complexes and to bind streoselec-
tively, enhances their performance for the chiral resolution
of enantiomeric species in both HPLC and CE. The use of
the CDs as the chiral selector was pioneered by Fanali.8
Since that report there has been a spectacular growth in the
use of native9 (a, b and g) CDs and alkyl modified8,10-16
CDs in CE. Among the various non-charged derivatives b
CD are versatile chiral selectors for the enantioseparation
of wide range of racemates. Cyclodextrins (CD) are well
known for potential to increase the apparent aqueous solu-
bility separation of low polarity compounds;17 they can be
added to CE separation buffers to increase the selectivity
and efficiency of CE separations.18
* Corresponding author. E-mail: malik_chem2002@yahoo.co.uk

1164 J. Chin. Chem. Soc., Vol. 56, No. 6, 2009
Malik et al.
Zhu et al17 have used dual cyclodextrin20 systems for
the chiral separation of the several drugs. Partial filling21 of
the capillary with the CDs for coupling CE with MS has
also been reported. There have been an increasing number
of applications of CZE to the analysis of pollutants in envi-
ronmental samples. Pesticides, a case in point, are of great
environmental concerns because of their high toxicities
even at low concentrations and are in common use.
electrophoretic system equipped with diode array detector.
The fused silica capillaries (37 cm total length, 30 cm de-
tector length) with internal diameter of 50 mm were used in
the setup. The transformation in effective mobility and the
integration of the peak areas for quantification was achieved
by gel-treat-software.23 All pH were adjusted with a 716
DMS Titrino equipped with a 6.0234.100 pH electrode
from Metrohm (Herisau, Switzerland).
The discovery of the phenoxyacetic acid herbicides
during the Second World War heralded the modern era of
agrochemicals. The fact that they are still being used today,
more than fifty years later is a testament to their efficacy,
safety and low cost. These compounds work by imitating
the natural plant growth hormone, indole-3-acetic acid
which causes abnormal growth which results in plant death.
The phenoxyacetic acids are therefore often referred as the
hormone or auxin herbicides.
Chemicals and reagent
Highly sulphated cyclodextrins were obtained as
20% (w/v) aqueous solutions from Beckman coulter Inc
(Fullerton, Ca, USA). Dicloprop, fenoprop, mecoprop and
2,4-DB (Fig. 1) were obtained from Dr. Ehrenstrofer GmbH,
Augsburg and 1,3,6,8-Pyrenetetrasulfonate (PTS) was also
obtained from Beckman.
HSCD solution was made in the buffers and they
were found to be highly soluble. All the solutions of the
phexoxy acid herbicides were prepared by dissolving 2 mg
of each in 1 mL pesticide grade methanol this was diluted
with 1 mL distilled water and further dilutions were made
as desired.
In this paper we described chiral separation of phen-
oxyacid using HSCD22 in the back ground electrolyte. The
sulphated group on the cyclodextrin gives derivative which
is negatively charged and highly soluble in aqueous solu-
tions. In order to test the effectiveness of HSCD as the
chiral selector, we have targeted two classes of the race-
mates. The first is the phenoxy acid herbicides and the sec-
ond class of racemate is 1-(1-aminoethyl)naphthalene.
Capillary conditioning
All new capillaries were pretreated with a 0.1 mol L–1
sodium hydroxide for 40 min. The capillary was condi-
tioned at the beginning of each day with 0.1 mol L–1 sodium
hydroxide for 30 min and 5 min before each run using high
pressure (20 psig; 1 psig = 6894.76 Pa) and between the
runs the capillary was rinsed for 5 min.
EXPERIMENTAL
Instrumentation
Separation and the analysis were performed with
PACE 5510 (Beckman, Fullerton, CA, USA) capillary
Procedure
Buffer vials containing the HSCD were placed in
Table 1. The working condition for CE
Capillary
Detection
Uncoated fused-silica, inner diameter: 50 mm,
effective length: 30 cm, total length: 37 cm.
210 nm
Detector cell size: 100*800 mm.
Cartridge set to 30 oC.
Temperature
Capillary preconditioning
Injection
2 min pressure with 0.1 mol/L NaOH, then 4 min pressure with BGE.
Hydrodynamic injection of sample 50 mbar for 10 s
(injected volume 58 nl).
Separation voltage
Typical current
Replenishment
10-20 kV.
~100 mA.
Replenishment of both separation vials and injection vial is preferred
after each injection.
Capillary preconditioning
(new capillary only)
Storing of capillary
Pressure flush with 0.1 mol/L NaOH for 30 min followed by BGE for
15 min.
Pressure flush with 0.1 mol/L NaOH for 15 min followed by milli Q
water for 15 min then with air for 5 min.
(more then one week)

Enantiomer Separation of Phenoxy Acid Herbicides by CE
J. Chin. Chem. Soc., Vol. 56, No. 6, 2009 1165
Fig. 1. Structures of phenoxy acid herbicides, 1-(1-aminoethyl)naphthalene and a-HSCD.
both the outlet and the inlet vial. The CE runs were per-
formed with reverse polarity (anode in the outlet vial) using
acidic ammoniumformate buffer (20 mmol, pH = 3). Under
these conditions of suppressed electroendoosmatic flow
(EOF), the analytes are mobilized to the anode by com-
plexation with the migrating negatively charged sulphated
cyclodextrin. PTS was taken as the reference marker.
to effectively interact with the two enantiomers.17 At pH =
3, 1-(1-aminoethyl)naphthalene will be in the form of
quartinary salt, i.e., having a charge on the nitrogen moiety.
This positively charged species can interact with the –OH
group of the cyclodextrins through hydrogen bonding or
-
otherwise this R-NH3+ can interact with the –OSO3 moiety
of cyclodextrins through electrostatic interactions.17
Optimizations were also made for the separation of
PAH. It was found that though a-HSCD (Fig. 1) was effec-
tive for the chiral resolution of mecoprop and dicloprop,
failed for fenoprop Fig. 3. With b-HSCD in the background
RESULTS AND DISCUSSION
The objective of using HSCDs for CE based chiral
separation is to resolve enantiomeric species. The sul-
phated moieties in HSCDs act as the charge separation car-
riers for the electrophoretic separation while the host guest
molecular interaction should remain essentially intact in
the hydrophobic pocket. Chen22 et al investigated the de-
gree of sulphonation of CD for the chiral resolution of the
neutral racemate glutethimide. They observed an increased
resolution with increase in the degree of sulphonation at the
expense of the analysis time. The cyclodextrins used here
were obtained from Beckman coulter with an average num-
ber of 11 sulphates for the a-CD, 12 for the b-CD and 13
for the g-CD.
Fig. 2. Electropherogram obtained for the enantiomer
separation of 1-(1-aminoethyl)naphthalene us-
ing ammonium formate buffer (pH 3, conc 25
mM, 5% b-HSCD, 15 kV, capillary length: 30/
37 cm, i.d. 50 mm), wavelength 221, reverse po-
larity. 1,3,6,8-Pyrenetetrasulfonate (PTS) is
used as an EOF marker.
Baseline separation of chiral 1-(1-aminoethyl)naph-
thalene was achieved using ammonium formate buffer (pH
= 3, conc = 25 mmol, capillary length = 30/37, i.d = 50 mm)
Fig. 2. High resolution is attributed to ability of the HSCD

1166 J. Chin. Chem. Soc., Vol. 56, No. 6, 2009
Malik et al.
electrolyte a resolution of R¢ = 93 for fenoprop and R¢ =
100 for dicloprop and mecoprop (R¢ = 100(H-H’)/H, where
H and H’ are the height of the first peak and the valley be-
tween the peaks)20 was achieved (Fig. 4). Effect of the in-
crease in HSCD concentration in the background buffer on
the resolution of the fenoprop is shown in Fig. 5. Decrease
in the migration time of the fenoprop with the increase in
the concentration of HSCD in the buffer is due to the in-
crease in the current and hence higher mobility. Fenoprop
is found to move faster than dicloprop with b-HSCD in
background electrolyte (Fig. 4) than with a-HSCD in back-
ground electrolyte (Fig. 3). This behavior is dependent on
the size of the molecules. The size restriction is logical,
since the usual complexation mechanism involves inser-
tion of the more hydrophobic (usually the aromatic) por-
tion of the molecule into the hydrophobic CD cavity.
At the experimental condition i.e. pH = 3, the phen-
oxyacid herbicides mentioned will exist in their partial dis-
sociated form and will be having negative charge on the
carboxyl group. This negative charge species can undergo
hydrogen bonding with the hydroxyl group of cyclodex-
trins moiety. The benzene ring of the PAH will rest in the
hydrophobic pocket of the HSCD.
CONCLUSION
HSCD namely a-HSCD, b-HSCD and g-HSCD have
been tried as chiral additives in the CZE separation of the
enantiomers of the chiral herbicides mecoprop and diclo-
prop, fenoprop. Fenoprop, which has the largest effective
PTS
25
2 2'
3
20
15
10
5
1 1'
(a)
20
15
10
5
0
0
2
4
6
8
10
Tim e (m in)
0
Fig. 3. Electropherogram obtained for the phenoxy
acid herbicides dicloprop (1, 1¢), mecoprop (2,
2¢), fenoprop (3), showing mutual as well as the
chiral separation (ammonium formate buffer
(20 mM, pH 3, 5% a-HSCD, 15 kV at 210 nm,
reverse polarity), capillary length: 30/37 cm,
i.d. 50 mm, reverse polarity. 1,3,6,8-Pyrene-
tetrasulfonate (PTS) is used as an EOF marker.
15
16
17
18
19
20
Time (min)
(b)
20
15
10
5
0
15
16
17
18
19
20
Time (min)
(c)
20
15
10
5
0
Fig. 4. Electropherograms obtained for the phenoxy
acid herbicides of fenoprop (1, 1¢), dicloprop
(2, 2¢), mecoprop (3, 3¢) and 2,4-DB (4) show-
ing mutual as well as the chiral separation.
Background electrolyte: ammonium formate
buffer (20 mM, pH 3), 6% b-HSCD, capillary
length: 30/37 cm, i.d. 50 mm, 10 kV at 210 nm,
reverse polarity. 1,3,6,8-Pyrenetetrasulfonate
(PTS) is used as an EOF marker.
15
16
17
18
19
20
Time (min)
Fig. 5. Electropherograms showing the separation of
fenoprop as a function of b-HSCD concentra-
tion (a) 3.3% b-HSCD (b) 5% b-HSCD (c) 6%
b-HSCD using ammonium formate buffer (pH
3, 20 mM), capillary length: 30/37 cm, i.d. 50
mm, wavelength 210 nm, reverse polarity.

Enantiomer Separation of Phenoxy Acid Herbicides by CE
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