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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M bil h dditi
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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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M bil h dditi
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Mobile phase additivesEffects-2
n-heptane/ EtOH/AcOH
(60:40:0.5)
n- heptane/ EtOH/TFA
(60:40:0.5)
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M bil h dditi
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Mobile phase additivesEffects-3
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