practical chiral hplc and

Method developMent
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Wide variety of chiral columns available.
Commercialized chiral columns:>200 Types
Most enantiomers can be readily separable
Method 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 enantiomers
Obtain 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(final
drugs or intermediates)at
production scale
5kg to 100
tons
Until industrial scale production
mg to 10 kg
Chiral chromatography
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Enantiomeric separation:
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Resolution formula=
(t2-t1)/0.5(tw2-tw1)
Enantiomeric resolution:
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Enantiomeric purity= Major/(Major + Minor)
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0
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 phases
Mixed 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 backbone
Steric 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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Available 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.

Available 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 V2 Chirobiotic T and T2
Available columns:
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Mobile phases for coated CSPs:
Normal Phase conditions: Polar mode:
Alkane/IPA
Alkane/Ethanol
Acetonitrile
Ethanol
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
Acetone
DMF
THF
DMSO
Never use
(even as a sample solvent)
These will irreversibly
destroy the coated CSP
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Before connecting the column 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 CSPs
Elution order
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Examples
Alkane/Alcohols
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Examples
Alkane/Alcohols
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Method development on CHIRALPAK IA
Effects of solvent modifiers
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Method development on CHIRALPAK IA
Effects of solvent modifiers
Important selectivity
Change can be
Observed with different
Solvent modifiers
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Mobile phase additives
Amines, acids, salts
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Mobile phase additives
Effects-1
Hexane/2-IPA/ Diethylamine
(90:10:0.1)
Hexane/2-IPA
(90:10)
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Mobile phase additives
Effects-2
n-heptane/ EtOH/AcOH
(60:40:0.5)
n- heptane/ EtOH/TFA
(60:40:0.5)
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Mobile phase additives
Effects-3
ACN 0.1%DEA
ACN 0.1% Butylamine
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UV cut-off of common HPLC solvents
Feasibility 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, chirobiotic
Amino Acids Cyclodextrins,Protein,Chirobiotic
Amines Protein, celulose/Amylose,Pirkle, chirobiotic,Cyclodextrin,Chirobiotic
Alcohols Protein, celulose/Amylose,Pirkle, chirobiotic,Cyclodextrin
Amides Protein, celulose/Amylose,Pirkle,Cyclodextrin,Chirobiotic
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 Protein
Aromatic drugs Protein, Pirkle
Lactones Celulose, Pirkle
Cyclic ketones Pirkle,Celulose
Alkaloids Celulose,Pirkle
Dihydro pyridines Celulose, Pirkle
NSAIDS Pirkle,celulose,Protein
Oxazolindones Pirkle
Peptides Chirobiotic
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Questions???
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Terima Kasih