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)

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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

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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

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

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