Chiral HPLC

8/12/2019 Chiral HPLC

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pr ctic l chir l hplc ndMethod developMent

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Wide variety of chiral columns available.

Commercialized chiral columns:>200 Types

Most enantiomers can be readily separableMethod can be readily validated.

Method can be readily scaled up to

commercial production.

Chiral chromatography?

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Early stages of development:

Fastest and more economical way to:

Analyze chiral compounds

Isolate individual enantiomersObtain the two enantiomers for

clinical toxicological studies

Scale up from bench- to pilot plant scale

Full development / production

Highly competitive way to:

Isolate single enantiomers(finaldrugs or intermediates)at

production scale

5kg to 100

tons

Until industrial scale production

mg to 10 kg

Chiral

chromatography

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Enant iomeric separat ion :

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Resolution formula= (t2-t1)/0.5(tw2-tw1)

Enantiomeric resolution:

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Enantiomeric purity= Major/(Major + Minor) 100

Enantiomeric purity and

enantiomeric excess(ee):

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Types of CSP..!

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Types of CSPs and their loading capacities:

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Which type of CSP?

1999 2001 2003 2005

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Small chiral molecules bonded to silica.

More specific applications; strong 3-point interactions through 3-classes:

-donor phases

-acceptor phasesMixed donor-acceptor phases

Binding sites are -basic or -acidic aromatic rings(-interactions),

acidic and basic sites(H-bonding), and steric interaction.

Mostly used with normal phase HPLC , SFC(super critical fluid chrom).

Limitations: Only works with aromatic compounds.

Available columns: WhelkO1, Whelk O2,ULMO (mixed phases) etc.

A= acidic group

B= acidic site

C= basic site

Brush type csp (3 point interaction model):

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Polymer based carbohydrates:

Chiral polysaccharide derivatives, i.e.; amylose and cellulose coated on a

silica support.

Enantiomers form a hydrogen bonds with carbamate links b/w side chain

and polysaccharide backboneSteric restrictions at polysaccharide backbone may prevent access of one of

Enantiomers to H-bonding site.

Can be used with normal phase HPLC, SFC,RP-HPLC

Limitations: Not compatible with a wide range of solvents other than alcohols.

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Avai lab le columns:

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Cyclodextrin Based CSPs:

Alpha, Beta and Gamma-cyclodextrin bond to silica

and form chiral cavities

Three points interaction by:

1. Opening of cyclodextrin cavity contains hydroxyls

for hydrogen bonding with polar groups of analyte.

2. Hydrophobic part of analyte fits into non polar

cavity (inclusion complexes)

One enantiomer will be able to better fit in the cavity than the other.

Used in RP-HPLC and polar organic mode.

Limitation:Analyte must have hydrophobic or aromatic group to fit into cavity.

Avai lab le columns:

Cyclobond (Alpha, Beta and Gamma cyclodextrins) from Astec. Inc.

ORpak CDA(Alpha), ORpak CDB(Beta), ORpak(Gamma) from JM Sciences.

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Chirobiotic phases:

Macrocyclic glycopeptides linked to silica

Contain a large number of chiral centers together

with cavities for analytes to enter and interact.

Potentials interactions:-complexes, H-bonding, ionic interactions.

Inclusion complexation, steric interactions.

Capable of running RP-HPLC, normal phase,

polar organic and polar ionic modes

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Chirobiotic v and V2Chirobiotic T and T2

Avai lab le columns :

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Mobile phases for coated CSPs:Normal Phase conditions: Polar mode:

Alkane/IPAAlkane/Ethanol

AcetonitrileEthanol

Methanol

Other alcohols

Reversed Phase conditions (RH-Versions):

Water/Alcohol or Acetonitrile

Phosphate buffer(pH 2-8)/Alcohol or Acetonitrile

KPF6 pH 2/ Acetonitrile with chiralcel OD-RH

Borate buffer (pH 9) Alcohol or Acetonitrile with chiral pak AD-RH

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Mobile phases for coated CSPs:

Really! coated CSPs are not stable with all solvents

Chloroform

Ethyl acetate

DCM

AcetoneDMF

THF

DMSO

Never use

(even as a sample solvent)

These will irreversibly

destroy the coated CSP

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Before connect ing the colum n to the system:

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Background

Coated polysaccharide-derived CSPs:

Highly selective

Broad application domain

High loading capacity

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Solvent compatibility

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Method development.!

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Screening approach

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Method development on ChiralpakIA

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Applications of coated and immobilized CSPs:Non-standard mobile phase

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Three immobilised CSPsElution order

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ExamplesAlkane/Alcohols

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ExamplesAlkane/Alcohols

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Method development on CHIRALPAK IAEffects of solvent modifiers

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Method development on CHIRALPAK IAEffects of solvent modifiers

Important selectivityChange can be

Observed with different

Solvent modifiers

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Mobile phase additivesAmines, acids, salts

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Mobile phase additivesEffects-1

Hexane/2-IPA/ Diethylamine

(90:10:0.1)

Hexane/2-IPA

(90:10)

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n-heptane/ EtOH/AcOH

(60:40:0.5)

n- heptane/ EtOH/TFA

(60:40:0.5)

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ACN 0.1%DEA

ACN 0.1% Butylamine 39


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UV cut-off of common HPLC solventsFeasibility of UV detection

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Regeneration in practice

Procedures:

Purpose:

Recover the specific supra molecular structure

Ensure the consistency of the column performance

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Class of compound Types of CSP

Acids Protein, celulose/Amylose,Pirkle, chirobioticAmino Acids Cyclodextrins,Protein,Chirobiotic

Amines Protein, celulose/Amylose,Pirkle, chirobiotic,Cyclodext

Alcohols Protein, celulose/Amylose,Pirkle, chirobiotic,Cyclodext

Amides Protein, celulose/Amylose,Pirkle,Cyclodextrin,Chirobiot

Esters celulose,Pirkle, chirobiotic.

Sulfoxides celulose,Pirkle, chirobiotic,Protein.

Carbamates Pirkle

Ureas Pirkle

Crown ethers Cyclodextrins.Metallocenes Cyclodextrins.

Thiols Pirkle

Amino alcohols Pirkle,Chirobiotic

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Class of compound Types of CSP

Succinamides Pirkle.

Hydantoins Pirkle,Chirobiotic

Bi-napthol Pirkle

-Lactams Parkle,Celulose

Polycyclic aromatic hydrocarbons Cyclodextrins

Cyclic drugs ProteinAromatic drugs Protein, Pirkle

Lactones Celulose, Pirkle

Cyclic ketones Pirkle,Celulose

Alkaloids Celulose,PirkleDihydro pyridines Celulose, Pirkle

NSAIDS Pirkle,celulose,Protein

Oxazolindones Pirkle

Peptides Chirobiotic43


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Questions???

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Terima Kasih

Documents

Chiral HPLC