Chiral chromatography & ion pair chromatography

It contain types , Principle and application of Chiral chromatography & ion pair chromatography

1. welcome
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2. CHIRAL CHROMATOGRAPHY
AND
ION-PAIR CHROMATOGRAPHY
PrsentedBy
Prof. HemantBansode
Assistant Prof.
DattakalaCollege of Pharmacy
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3. Chiral Chromatography
Content-
Introduction.
Principle.
Applications.
Ion-Pair Chromatography
Content-
Introduction.
Principle.
factors affecting IPC.
Advantages.
Applications.
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4. Stereochemistry Terms
•Isomers:
Compounds with the different chemical structures and the same molecular
formula
•Stereoisomers:
compounds made up of the same atoms but have different arrangement of
atoms in space
•Enantiomers are the 2 mirror image forms of a chiralmolecule
–can contain any number of chiralcenters, as long as each center is the
exact mirror image of the corresponding center in the other molecule
–Identical physical and chemical properties, but may have different
biological profiles. Need chiralrecognition to be separated.
–Different optical rotations (One enantiomer is (+) or dextrorotatory
(clockwise), while the other is (-) or levorotatory (counter clockwise))
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5. •Racemate:
•A 1:1 mixture of enantiomers
•Separation of enantiomers occurs when mixture is reacted with a chiralstationary
phase to form 2 diastereomeric complexes that can be separated by chromatographic
techniques.
•Diastereomers:
•stereoisomers that are not enantiomers Have different chemical and physical
characteristics, and can be separated by non-chiral methods.
•Has at least 2 chiralcenters; the number of potential diastereomers for each chiral
center is determined by the equation 2
n
, where n=the number of chiralcenters
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6. Relationships of Stereoisomers
Conformational isomers
Isomers: Compounds with the
same molecular formula
Constitutional (or structural)
isomers
Stereoisomers
Same atom
connectivity
Different atom
connectivity
Interconvert through
rotation about a
single bond
Conformational
isomers or rotamers
Configurational
isomers
Not readily
Interconvertible
EnantiomersDiastereomers
Chiral
w/
chiralcenters (optically active)
w/o
chiralcenters (opt. inactive)
Geometric isomers
Achiral
Configurational isomers
mirror images
Enantiomers
Cis, Trans
(E,Z) isomers
cis and trans isomers
Constitutional (structural)
isomers
mirror images at this carbon
Enantiomeric
Not mirror images
Diastereomers
Not mirror images at this carbon
Diastereomeric
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7. Chiral chromatography
•Chiral molecule:-
Is defined as a molecule that is not superimposible on its mirror
image.
•The two mirror image forms of a chiralmolecule are……….
Enantiomers ,
Dienantiomer.
•A chiralmolecule has one or more stereogenic centers which are typically carbons.
These stereogenic carbon atom are attached to four different substitute.
Example, Chlorobromomethanol.
•Based on direction of optical rotationthese are …………….
Dextro (+) isomer,
Levo (-) isomer.
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8. •Each enantiomer has an equal but opposite optical rotation; can be measured
using optical rotation polarimeter
•One enantiomer rotates polarized light in a clockwise direction and is then designed
as (+), or dextrorotatory
•The other enantiomer rotates polarized light in counter-clockwise direction and is the
(-) enantiomer, or levorotatory
•Racemates (1:1 mixture of enantiomers) have no observable optical rotation; they
cancel each other out
Determination of Optical Activity:-
Specific Rotation = []
D=

l * c
where
= observed rotation,
l = cell length in dm,
c = concentration in g/mL, and D is the 589nm light from a sodium lamp.
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9. Chiral vs Achiral Compounds
Achiral Molecule:
•Has no stereogenic center;
•has a plane of symmetry
•one that issuperimposable on its
mirror image (the two are
identical)
–i.e. nail, ball, a baseball bat
•Not optically active
Chiral Molecule:
•Has one stereogenic center
•one that is notsuperimposable on
its mirror image (the two are not
identical)
–i.e. hands, keys, shoes
•the two mirror image forms are
called enantiomers
•Optically active
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11. Chiral Chromatography
•Separation of enantiomer is most rapidly accomplished by means of chiral
chromatograph.
•Chiral chromatography include the use of…
1. Gas chromatography (GC)
2. Supercritical fluid chromatography(SFC)
3. Capillary electrophoresis(CE).
4. Chiral HPLC.
•The seperation of two enantiomers is measured by Enantioselectivity (α).
•A pair of enantiomers is considered to be resolve if α> 1.1
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12. Principle:
•The sample is reacted with chiralderivatizing agent or ineract enantiomers with a
chiralphase so as to form diasteromers or diastereomeric complexes .
•Diastereomers have different chemical and physical properties and can be seperated
by HPLC.
•Indirect method of chiralanalysis uses chiralstationary phases(CSP) along with
derivatiziung agents.
•The use of CSP chiralstationary phases without derivatizing agent called as direct
method of analysis.
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14. •Single enantiomersof chiralactive pharmaceutical
ingredients (APIs) may have different:
–Pharmacokinetic properties in animal models
•Absorption, distribution, metabolism and excretion
–Pharmacological or toxicological effects
•Biologically “active”isomer may have desirable effects
•Biologically “inactive”isomer may have undesirable side effects (i.e.
increased toxicity)
•Increased pressures by regulatory authorities to
switch from racemicto single enantiomerAPIs
•Development of chiralAPIs raises issues regarding:
–acceptable manufacturing control of synthesis and impurities
–pharmacological and toxicological assessment of both
enantiomers
–proper assessment of metabolism and distribution
–proper clinical evaluation of these drugs Racemate vs. Single Enantiomer
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15. •Chiral Derivatization :
•It involves reaction of an enantiomeric molecule with an enantiomerically
chiral derivatizing agent (CDA) to form two diasteriomeric derivatives.
•These can be separated by using ……
Conventional normal phase HPLC
Reversed phase HPLC
•Normal phase HPLC has …
better selectivity for Isomers,
no racemization ,
Should be optically pure (>90%)
•Example, ,
Resolution of ±Naproxen by using L-alanine-ß-napthylamide as
CDA.
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16. Chiral stationary phase :-
•Chiral substance that is chemically bonded or coated to a stationary phase
support to form a chiralstationary phase(CSP).
•CSP interacts with analyte enantiomers to form short-lived transient diastereomeric
complex.
•Silica or aminopropyl silica is commonly used as starting support material.
•The CSP can be attached to the support in various ways;
Covalently bonded ,
Ionically bonded,
Physically bonded.
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17. Dattakala College of Pharmacy
Classification of Chiral Stationary
Phases (CSP)
1)Polymer-based Carbohydrates
–Chiral polysaccharide derivatives, i.e. amylose and cellulose, coated on a
silica support.
–Enantiomers form H-bonds with carbamate links between side chains 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.
Available columns
i.e. Chiralpak AD, AD-RH, AS, AS-RH, and Chiralcel OD, OD-RH, OJ, OJ-
RH, etc. Chiral Technologies, Inc.
-
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18. •Polysaccharide (Carbohydrate) Columns:
•Cellulose and amylose derivative,
•These columns are extensively used for both analysis and the preparative
separations of wide range of enantiomers.
•Properties of commercial polysaccharides phases:
•High sample loading capacity ,
•Facilitating preparative isolation ,
•Typically use readily removed organic mobile phases ,Simplifying isolation,
•Broadly applicable to wide range of compound types.
•a) Microcrystalline cellulose Triacetate (MCT),
•b) Cellulose and Amylose –based column pickings .
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19. Classification of Chiral Stationary
Phases (CSP)
2)Pirkle or Brush-type Phases: (Donor-Acceptor)
–Small chiral molecules bonded to silica
–More specific applications; strong 3-point interactions through 3 classes:
•π-donor phases
•π-acceptor phases
•Mixed donor-acceptor phases
–Binding sites are π-basic or π-acidic aromatic rings (π-πinteractions),
acidic and basic sites (H-bonding), and steric interaction
–Separation occurs through preferential binding of one enantiomer to CSP
–Mostly used with normal phase HPLC, SFC.
–May get less resolution with RP-HPLC; compatible with a broad range of
solvents,
–Limitations: only works with aromatic compounds
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20. Classification of Chiral Stationary
Phases (CSP)
3)Cyclodextrin CSPs
–Alpha, beta and gamma-cyclodextrins bond to silica and form chiral cavities
–3-point interactions by:
•Opening of cyclodextrin cavity contains hydroxyls for H-bonding with
polar groups of analyte
•Hydrophobic portion 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
–Limitations: analyte must have hydrophobic or aromatic group to “fit” into
cavity
•Available columns:
-Cyclobond (-, -, and -cyclodextrins) from Astec, Inc.
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21. Classification of Chiral Stationary
Phases (CSP)
4)Chirobiotic Phases
–Macrocyclic glycopeptides linked to silica
–Contain a large number of chiral centers together with cavities for
analytes to enter and interact
–Potential interactions:
•π-πcomplexes, H-bonding, ionic interactions
•Inclusion complexation, steric interactions
–Capable of running in RP-HPLC, normal phase, polar organic, and
polar ionic modes
•Available columns:
–Chirobiotic V and V2 (Vancomycin), Chirobiotic T and T2
(Teicoplanin), Chirobiotic R (Ristocetin A) from Astec
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22. Classification of Chiral Stationary
Phases (CSP)
5)Protein-based CSPs:-
–Natural proteins bonded to a silica matrix
–Proteins contain large numbers of chiralcenters and interact
strongly with small chiralanalytes through:
•Hydrophobic and electrostatic interactions, H-bonding
–Limitations:
•Requires aqueous based conditions in RP-HPLC
•Analyte must have ionizable groups such as amine or acid.
•Not suited for preparative applications due to low sample
capacity
•Available columns:
-Chiral AGP (-glycoprotein) from ChromTech
-HSA (human serum albumin) from ChromTech
-BSA (bovine serum albumin) from Regis Technologies
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23. Protein derived chiral stationary phases:
•Following are the six materils with different charactaristics, commercialized in
protein phase columns for chiral separation:
1) BSA: Bovine serum albamin
2) HAS: Human serum albumin,
3) AGP: α
1 acid glycoprotein (orosomucoid),
4) OVM: ovomucoid,
5) CBH : Cellobiohydrolase,
6) Pepsin
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24. •These protein have been covalently bonded to wide pore silica and polymeric
supports for rapid HPLC separations that are suited for routine analytical method.
•Special advantage of these column is that they are compatible with the aqueous
buffered mobile phases widely used in many biological application.
Charactaristics of protein based chiralcolumns:
Sr.N
o.
ProteinMol.wt.Mol.
S-S bridges
Parent
carboh-
ydrate
Support Partical
size
Pore
size
01BSA
HSA
66,000 17 _ Silica 7 & 5100-
300
02AGP 141,000 2 45 Silica 5 &10120-
250
03OVM 28,800 8 30 Silica /
Polymer
5 & 15-
20
120-
500
04CBH 64,000 12 6 Silica 5 120
05Pepsin 34,600 3 _ Silica 5 120
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25. Sr.No.Type of CSP Chiral recognition
mechanism
Analyte
Requirment
Mobile phase
Requirment
01 Protein based Hydrophobicand
Electrostatic
interactions
Ionizable group
(amine/acid)
Aromatic group
Reversed phase
only
02 Cyclodextrine Inclusion
complexation,
Hydrogen bonding
Polar and aromatic
group
Reversed and
normal
phase,
Polar organic
mode
03 Polymer based
carbohydrate
Inclusion
complexation
attractive interaction
Ability to H-
bonding,terics bulk
near chiralcentre .
Reversed phase
and normal
phase
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26. Characteristic of sample for chiralHPLC
•Solubilityin different solvents,
•Πor Hydrogen bonding capacity,
•Functional group,
•UV spectrum,
•Bulky substituents,
•Inclusion complexingcapability,
•The arrangment in space of these substituent groups plays an important role in
enantiomer separations.
•The closer a group is to the chiralcentre the more likely is chiralrecognition and
enantioselectivity.
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•Over 100 column are commercially available.
•Many of these column similar in structure and enantioselectivity.
•A very rough order of CSP universality is…
Protein > Carbohydrete> Pirke> Cyclodextrin .

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Application:
Separation of enantiomers.
Resolution of wide range of neutral , basic and acidic drug obtained from nature,
synthesis or biological origin.
For racemate separation.

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ION-PAIR
CHROMATOGRAPHY

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ION –PAIR CHROMATOGRAPHY :
It is modification of cation / anion exchange chromatography.
In HPLC technique used for separation of charged analytes in which formation of
ion pair takes place with addition of counter ion and then this neutral ion pair
complex Undergoes partitioning between stationary and mobile phase.
Can be perform normal phase and reverse phase chromatography.

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Mechanism of Ion-Pair chromatographic retention:
Ion-pair formation.
Dynamic ion exchange.
Ion interaction model.

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Ion pair reagents:
It is a large organic counter ion which is added to mobile phase at low concentration.
It self is charged and used to separate organic solute ions of opposite charge by
forming ion pair complex by above proposed mechanisms.
1) for basic compound:-
separated by addition of HCL , Perchloric acid , Perfluorocarboxylic acid sulphoric
acid to the mobile phase.
Ph of mobile phase 3-4.
2) For Acidic compound:-
Alkyl quaternary ammonium salts or Alkyl amines.
Ph of mobile phase 7.5

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Factor affecting Ion pair chromatography:
1) Ph :
Strength of association of ion pair is function of Ph.
2)Size of lipophilic group on counter ion(ion pair reagent):
It affect on degree of retention.
3)Concentration of counter ion:
Increase the concentration of counter ion there is increase in retention time.

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4) Organic modifier:
These are used to decrease retention times and to modify selectivity of separation.
This work as following:-
a)By competing with pairing reagent for stationary phase.
b)By decreasing polarity of mobile phase this affect partitioning of analyte –reagent
pair in to hydrophobic environment.
Methanol & Acetonititrile are commonly used organic modifier.
5) Temperature:
Change in temperature causes variation in degree of ionization.
Ion –pair chromatography is carried out at room temperature or much lower
temperature less than 20
0
c

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Advantages of ion-Pairing:-
Suitable for simultaneous analysis of non ionic and ionic species.
Ion –pair formation is relatively faster.
It is carried out at room temperature.
Allows sensitivityto be manipulated through change in composition of mobile
phase alone.
Does not require special stationary phase or equipment.
These provide wide choice of additives to improve separation.
Gives sharp peak and highly reproducible results.
Revere phase column provide higher efficiency and greater flexibility.
Biological sample can be separated.

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Application of ion –pair chromatography:-
Determination of surfactants and similar analytes.
Organic cations like aliphatic and aromatic can be separated.
Screening of beta blockers in human serum.
Separation of biological amines.
DNA-RNA analysis.
SVERI's College of Pharmacy, Pandharpur
Dattakala College of Pharmacy

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References:
1] Pirkle W. H. and Pochapsky T .C., “Advances in chromatography’’ Giddings G.C,
Grushka E & Brown P. R.; Marcel Dekker inc. NY volume 27, 73-127 [ 1987.]
2] D. R Taylor & K. Maher, “ Chiral separation by high Performance liquid
chromatography,” J. Chromatography sci., 30, 67-85 ,[1992].
3] Fundamentals of analytical chemistry by Skoog, WEST , Holler, 991-92
Organic chemistry Seyhan N. Ege, 3
rd
Edition, 198-200 .

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