Applications of Chiral Supercritical Fluid Chromatography

2. Chiral compound
Is a type of molecule that lacks an internal plane of symmetry

3. Have non-superimposable
mirror image
Have identical physical
and chemical properties
They differ in their
optical activity
Enantiomers (or optical isomers)

4. Stereospecific
Resulting in differences in pharmacodynamics and/or pharmacokinetics (ADME)

5. One enantiomer may produce the
desired therapeutic action
While the other may be inactive or
even toxic

6. Since more than half of pharmaceutically-active ingredients are optically active, the industry
requires powerful means of chiral drug detection and separation

7. Chiral Resolution:
Is the separation of two enantiomers present in a racemic mixture or any mixture of
enantiomers
Chromatographic methods are the most popular, including:
 Gas chromatography (GC)
 Thin-layer chromatography (TLC)
 Capillary electrophoresis (CE)
 Capillary electrochromatography (CEC)
 Supercritical fluid chromatography (SFC)
 High-performance liquid chromatography (HPLC)

8. HPLC is the most widely used technique for the separation of enantiomers due to:
 Their extensive applications
 Ease of applicability
 Availability of advanced instrumentation
HPLC drawbacks:
 The use of large amounts of toxic solvents
 Potentially long equilibration and analysis times
 Significant peak broadening sometimes occurs as a result of the relatively slow diffusion

9. Supercritical fluid (SCF)
Any substance in a state above the critical temperature and critical pressure where liquids and gases can coexistis
 Has good solvating power
 High diffusion
Excellent choice as a mobile phase

10. They are considered as green mobile phases because of their:
Limited environmental impact
Reduced consumption of toxic solvents and additives
Lack of toxicity (in most cases)
Cost reduction of solvents and waste removal
Residue-free removal of the solvent from the extract

11. High speed Higher throughput
Smaller pressure
drop across the
column
Used with wide
range of sensitive
detectors
Smaller volumes of
toxic solvents and
hazardous waste
Higher column
efficiency
Improved
resolution
Extended
temperature
capability
Can be used for
analytical and
preparative-scales
Higher flow rates

12.  The temperature and pressure must be carefully controlled
 Expensive technology
 It is somewhat recent technique that still requires further developments
SFC is suitable
for non-polar
pharmaceuticals
Cannot be applied
to polar
compounds
This can be corrected by the addition of an organic modifier to the mobile phase e.g.
• Methanol
• Acetonitrile
• Methylene chloride

13. Three types of columns used:
 Capillary columns
 Open tubular columns
 Packed columns (most dominant)
Multiple chiral stationary phases (CSPs) and combinations of both
chiral and achiral stationary phases can be coupled in series in SFC
To achieve the desired selectivity

14.  Short columns (5 cm), which have potential advantages of:
 larger columns (250x 254 mm) for preparative analysis
 Traditional analytical-scale (250 mm x 4.6 mm) packed columns
Rapid method development
High-throughput applications

15. CO2 is the most commonly used for SFC for many reasons:
• Allows high flow rates
• Allows low pressure drop
Low viscosity
• Non toxic
• Non-flammable
• Readily available
• Inexpensive
Environment friendly
• Results in high efficiency separations
High diffusivity

16. Mobile phase parameters have an impact on
1) Pressure
 Enantioselective separations employ pressure that is above the critical pressure (P > PC)
 Has a greater impact on retention time than on enantioselectivity
 Its effect depends on the column used and the compounds being analyzed
Retention
time
Resolution Selectivity

17. Selectivity Temprature Selectivity
2) Temperature
 Enantioselective separations employ temperatures that are below the critical temperature (T < TC)
 Although column efficiency generally improves as temperature increases, but unusually:
Entropically driven Separations

18. 3) Flow rate
High
diffusivity
Low
viscosity
High
flow
rates

19. 3) Flow rate
Flow rate
Analysis time

20. 4) Modifiers
Methanol
Acetonitrile
2-propanol
Functions of
modifiers:
• Affect enantioselectivity
• May interact with the stationary phase and the analyte
• Impacts on the solvent strength
• May improve peak shape and enhance peak resolution

21. 5) Additives
 They are polar substances incorporated into the modifier at volume of 0.1–2.0%
 Usually, acidic additives are used for acidic compounds and basic additives for basic compounds
The most common additives used are: isopropylamine, triethylamine, acetic acid, and trifluoroacetic acid
Functions:
 Suppressing analyte ionization
 Masking active sites on the stationary phase
 Altering the polarity of the mobile phase

22. In chiral SFC, the separations are mainly performed by
Direct
approach
by chiral
selector
either
Bonded onto a
chromatographic
support
Added to the
mobile phase
using achiral
column
Chiral
stationary
phase
(CSP)
Simple
Effective
No need for
formation of
diastereomeric
derivatives

23. They can be classified into:
Such asMacromolecular
• Derivatized polysaccharides
• Proteins
• Synthetic polymers
Such asMacrocyclic
• Cyclodextrins
• Macrocyclic antibiotics
Such asLow molecular weight
• Pirkle-type CSPs

24. Of the many
polysaccharide-
based chiral
selectors
Cellulose and
Amylose are
recommended due
to:
• Their abundance
• Excellent capabilities
for chiral resolution
Even more
amylose provides
more chiral grooves
for enantiomeric
resolution
Applications:
• Determination of various
enantiomer–enantiomer
interactions
• Developing and
designing homochiral
drugs

25. Chiral recognition mechanism:
Enantiomer hydrogen bonding
Exists in helical conformation
Polysaccharide backbone
Steric effects
Side chain

26. Most commonly used because of:
 Enantioselectivity
 High loading capacity
 Their use in preparative scale
Chiralpak AD
Chiralpak AS
Chiralcel OD
Chiralcel OJ

27.  Homochiral, non-ionic, cyclic oligosaccharides
 Composed of 6–12 D-(+)-glucopyranose units connected through α-1, 4-linkages
 The most commonly used α-, β- and γ-cyclodextrins, containing 6, 7 and 8 glucopyranose units
Cyclodextrin
CSP causes
efficient
separation
Aminoglutethimide
by SFC
Thalidomide
by HPLC

28. Chiral recognition mechanism:
Inclusion of the bulky
hydrophobic group of
the analyte
Dipole-dipole interactions
& hydrogen bonds

29. Vancomycin Teicoplanin Ristocetin
Advantages
 They contain several stereogenic centers and functional groups
 Their ‘‘basket-like’’ geometry allows inclusion-type complexation
 They are amphoteric
 They are hydrophobic and hydrophilic in nature so, can be used in NP and RP modes

30. Chiral recognition mechanism:
Vancomycin

31. Advantages
 Used for wide variety of compound groups
 High coolumn durability
 Ability to invert elution order
 Used for analytical and preparative scales
 Universal solvent compatibility
Few applications of this CSP are reported for pharmaceutical-compound analysis
Chiral recognition mechanism:
 Electron donor-acceptor π-π interactions
 Hydrogen bonding
 Dipole-dipole interactions

32. Conditions of
synthesis
• Amount and type of
monomer employed
• Porogen
• Cross linker
• Temperature of
polymerization
Properties of
MIPs
• Stability
• Quality
• Porosity
• Accessibility of
recognition sites

33. Advantages
 Robust with high mechanical strength
 Resistant to elevated pressure or temperature
 Stable in the presence of extreme acids, bases or organic solvents
 Have special recognition sites with predetermined selectivity for the analyte (template)
Drawbacks
 Low chromatographic efficiency
 High peak asymmetry

34. Chiral recognition mechanism:

35. Powerful analytical and preparative technique for chiral
separations and purification e.g. caffeine and ketoconazole
Quantifying minor pollutants in chiral drugs
Can be used in the quality control of chiral pharmaceuticals
In-vivo analysis of chiral drugs and their metabolites e.g.
Warfarin, ketoprofen, propranolol
In forensic science, SFC is helpful for the identification of
synthesis or isolation sources of illicit materials e.g.
methamphetamine

36. Often provides faster
separations than HPLC
Easily transposed to the
preparative scale
Provides high flow rate due
to low viscosity
Cost, health and safety
benefits due to reduction in
the use of organic solvents
It has been demonstrated that SFC is rapidly replacing HPLC in many pharmaceutical situations
not only for purification but also as the standard screening and method development tool for chiral
compounds for the following reasons:

37. Future developments
Validation of the developed
methods leading to wider
use of the technique
Extended testing of
molecules to investigate the
enantioselectivity of a given
system