Separation of Chiral Pesticides by SFC and HPLC · 2014. 10. 6. · Separation of Chiral Pesticides...
Transcript of Separation of Chiral Pesticides by SFC and HPLC · 2014. 10. 6. · Separation of Chiral Pesticides...
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Separation of Chiral Pesticides
by SFC and HPLC
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Tong Zhang(1), Pilar Franco(1), Joseph Barendt(2)
(1) Chiral Technologies Europe (Illkirch, France)
(2) Chiral Technologies, Inc (PA 19380 USA)
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Chirality in Biologically Active Compounds
• Pharmaceuticals
• Flavor and Fragrance
• Agrochemicals
• Regulatory Guidance
– Varies by application and country
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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
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Optical Activity
• Identical physical and chemical properties
– In an isotropic environment
• Biological systems are distinctly anisotropic
– Uptake, bio-activity, metabolism affected
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Issues Specific to Chiral Compounds
• Efficacy of each isomer
• Metabolization rates may differ
• Biodegradation in environment
• Drives the need for chromatographic methods
– Analytical
– Preparative
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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
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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
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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)
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Chiral resolution of pesticide enantiomers
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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
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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)
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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)
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Chiral resolution of pesticide enantiomers by SFC Separation examples
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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
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Preparative Separations
• Why Preparative?
• Need for;
– Standard samples of each isomer
– Differential Toxicity testing
– Metabolism studies
– Biodegradation studies
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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
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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
*
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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
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Suitability of Scale
• SFC/HPLC
– Milligrams
– Grams
– Kilos
• Multi-Kilos and beyond - SMB
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Chromatography Equipment
SFC
HPLC
Batch and SMB
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Industrial Scale SMB
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Chiral Chromatography
• Effective technique for
– Analytical determination
– Preparative separation
– Lab scale (HPLC, SFC)
• mgs to kgs
– Commercial scale (HPLC, SMB)
• MTs
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THANK YOU!