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Separation of Chiral Pesticides
by SFC and HPLC
1
Tong Zhang(1), Pilar Franco(1), Joseph Barendt(2)
(1) Chiral Technologies Europe (Illkirch, France)
(2) Chiral Technologies, Inc (PA 19380 USA)
Chirality in Biologically Active Compounds
• Pharmaceuticals
• Flavor and Fragrance
• Agrochemicals
• Regulatory Guidance
– Varies by application and country
3
Chiral (28%)
Achiral organics (66%)
Biological (3%) Inorganic (3%)
470
1120
Pesticides: 1700 products
Insecticide (30%)
Herbicide (20%)
Fungicide (18%)
Acaricide (10%)
Plant growth regulator
Others
Insect attractant repellent
Rodencide Nematicide
Chiral pesticides: 470 products
Reference:
E.M. Ulrich et al. , Rev. Environ. Contam. Toxicol. 217 (2012) 1-74
Chirality in Pesticides
Optical Activity
• Identical physical and chemical properties
– In an isotropic environment
• Biological systems are distinctly anisotropic
– Uptake, bio-activity, metabolism affected
Issues Specific to Chiral Compounds
• Efficacy of each isomer
• Metabolization rates may differ
• Biodegradation in environment
• Drives the need for chromatographic methods
– Analytical
– Preparative
6
Polysaccharide-derived Chiral Stationary Phases
RO
O
OR
ORO n
RO
O
OR
OR
O
nAmylose-based Cellulose-based
-R CSP
CHIRALPAK IA NH
O CH3
CH3
CHIRALPAK IC
CHIRALPAK IE
CHIRALPAK IF
CHIRALPAK IB
CHIRALPAK ID
Polysaccharide
amylose
cellulose NH
O CH3
CH3
cellulose
amylose
amylose
amylose NH
O Cl
NH
O Cl
Cl
NH
O Cl
Cl
NH
O Cl
CH3
7
Experimental approaches Sample screening and effect of chiral selector
CHIRALPAK IC
CHIRALPAK ID
CHIRALPAK IE
CHIRALPAK IF
CHIRALPAK
CHIRALPAK IB
CHIRALPAK IA
Waste
pum
p
dete
ctio
n
N
N
Cl
Cl
OH
Fenarimol
(fungicide)
Common
Column: 150x4.6
EtOH/DEA 100:0.1
0.5 mL/min; 230nm
RO
O
OR
ORO n
Amylose-based
NH
O CH3
CH3
R:
RO
O
OR
OR
O
n
Cellulose-based
NH
O Cl
Cl
R:
RO
O
OR
ORO n
Amylose-based
NH
O Cl
ClR:
CHIRALPAK
CHIRALCEL
CHIRALCEL
Varying Modes
• HPLC
– Normal Phase
• Hexane/Alcohol, ACN, Alcohol, DCM, etc.
– Reversed Phase
• H2O/MeOH, H2O/ACN, H2O/THF, etc.
• SFC
– Supercritical CO2 plus modifier (~10% alcohol)
9
Chiral resolution of pesticide enantiomers
10
Common
CHIRALPAK IC-3 (150x4.6 mm i.d.)
Additive: 0.1% DEA
Bromuconazole
(fungicide)
O
NN
N
Cl
Cl
Br*
*
Enantio- and stereoselective resolution of pesticides
11
Effect of the mobile phase Online racemization and its inhibition
Common
CHIRALPAK IE-3 (150x4.6 mm i.d.)
DEA 0.1% in MP
NN
N
O
O
Cl
Triadimefon
(fungicide)
12
HPLC and SFC Complementary techniques
CH
IRA
LP
AK
ID
Benoxacor
(herbicide safener) CH
IRA
LP
AK
ID
O
N
OCl
Cl
min
CH
IRA
LP
AK
IC
-3
CH
IRA
LP
AK
IC
-3
N
OOO
Benalaxyl
(fungicide)
13
Chiral resolution of pesticide enantiomers by SFC Separation examples
14
Enantiomer resolution of pesticides Fast analysis by SFC by maximizing the kinetic performance of the column
BPr: 150 bar
Flow rate: 3.6 mL/min
Temperature: 40
C
UV Detection
System: ACQUITY UPC² TM
Column size: 100 x 3.0 mm i.d.
Mobile phase: CO2/MeOH 85:15 v/v
Additive:
(a) 1% DEA in MeOH;
(b) None
Time scale: 1 minute
Preparative Separations
• Why Preparative?
• Need for;
– Standard samples of each isomer
– Differential Toxicity testing
– Metabolism studies
– Biodegradation studies
Process For Prep Separation
• Similar to Analytical Method Development
– Matrix of columns/mobile phases
– Find best separations
• Optimize for maximum sample loading
– Larger separation between peaks
– Higher solubility in mobile phase
• Run “stacked injection” technique
Benzyl-3-methyl-4-piperidone
Analytical HPLC
Column : CHIRALPAK AD-H,
250 x 4.6 mm
Mobile Phase : Acetonitrile, 0.1%
DEA,
Flow Rate : 1 ml/min
Detection : UV, 245 nm
Sample : 10 g/l; 0.5 l
*
Benzyl-3-methyl-4-piperidone
150 ml/min
675 mg / injection
Acetonitrile
Cycle time 4 min
T = 25
C
10.1 g/hr
Column : CHIRALPAK AD
20 m, 500 x 50 mm
Suitability of Scale
• SFC/HPLC
– Milligrams
– Grams
– Kilos
• Multi-Kilos and beyond - SMB
Chromatography Equipment
SFC
HPLC
Batch and SMB
Industrial Scale SMB
Chiral Chromatography
• Effective technique for
– Analytical determination
– Preparative separation
– Lab scale (HPLC, SFC)
• mgs to kgs
– Commercial scale (HPLC, SMB)
• MTs
23
THANK YOU!