Separations of optical isomers requires interaction with
A chiral selector. Such a chiral selector can be present
In the stationary phase or in the mobile phase. The
preferred appraoch is one in which the chiral selector
Is bound to a solid stationary phase in a solid-liquid
System or distributed into a liquid in a liquid-liquid
The approaches to chiral separation are:
1. Ligand exchange – mobile phase additive; amino acids
2. Inclusion complex formation with cyclodextrins.
3. Interaction with a chiral selector based on 3 specific
Interactions on of which is strong and the other two are
Weak – 3 point contact model based on drug-receptor
4. Molecular imprinting – lock and key approach
Introduced by V. Davankov in the late 1960’s. Typically
Performed on a C18 column. The principle is:
CuL + D (or R) CuD + L; (KD)
CuL + L (or S) CuL + L; (KL)
The separation depends on the differences between KD
and KL and the differences in the capacity factors for
The complexes CuD and CuL between the mobile phase
And C18 stationary phase. The difference in the
Capacity factors is bound to be small. The chiral
Recognition occurs through differences in KD
and KL . This method does not yield pure analytes after
LIGAND EXCHANGE (CONTD)
L-proline is typically used as the exchanging ligand
INCLUSION COMPLEX FORMATION
CYCLODEXTRIN STATIONARY PHASES
THREE POINT CONTACT MODEL
C' A' C'
Chiral Recognition Molecule
MODES OF INTERACTION
1. π-π interaction.
2. Ion pair formation
3. Hydrogen bonding
4. Charge transfer interaction
5. Long range forces (van der Waal’s forces)
6. Steric interactions
Other antibiotics used: vancomycin
Problems assigned: All problems at the end
of the chapter (29-1 to 29-10).
Triple point is the temperature and pressure at which
the three phases solid, liquid, and gas coexist.
Supercritical point corresponds to the temperature
And pressure at which the distinction between liquid and
Gas does not exist.
Super critical temperature is the temperature above
which a distinct liquid phase does not exist. Vapor
Pressure of a substance at its critical temperature is its
At temperatures and pressures above the critical point
The substance is called a supercritical fluid.
PROPERTIES OF SUPECRITICAL FLUIDS
Properties of supercitical fluids are inermediate
Between its properties in the gaseous and liquid states.
Supercritical fluids have much lower surface tension
han their liquid form. This allows them spread across
urfaces very easily.
They maintain their ability to dissolve a wide range
f substances as in their liquid state.
Analytes dissolved in supercirtical fluids are easily
ecovered by lowering the pressure at relatively low temp.
PROPERTIES OF SUPECRITICAL FLUIDS
The supercritical pressure of CO2 at 32 C is 1070 psi and
At 49 C is 3500 psi. These pressures are well within
The pressures under which HPLC can be operated.
Supercritical CO2 can dissolve a variety of compounds
Such as n-alkanes (5 –30 C atoms), aromatics including
Polycyclic compounds, and di-n-alkylphthalates with
SFC is a hybrid of gas and liquid chromatography. It is
Useful for the separation of compounds that are not
Easily handled in gas or liquid chromatography.It
Is applicable for the following general cases:
1. Nonvolatile and thermally labile compounds that makes
Gas chromatography unsuitable.
2. Poor solubility in solvents employed in liquid
3. Compounds do not have functional groups that makes
Spectroscopic and electrochemical detection possible.
The column must be in a thermostated oven to achieve
The critical temperature. A restrictor or back pressure
Device is used to maintain the pressure at or above the
Analytes are detected by flame ionization.
Column: Open tubular and packed columns are employed.
Open tubular columns are fused silica columns with
Internal coatings of bonded and cross-linked siloxane
Polymers of various types.
Packed columns contain various packing materials
such as silica and polymeric beads.
Column length = 10 – 20 m; inner diameter = 0.05 –
0.1 mm; Film thickness for open tubular columns =
0.05 – 0.1 µm.
Packed columns contain particles of size 3 – 10 µm.
Column inner diameter can be 0.5 – 4.6 mm.
Particles can be normal or reverse phase or polymers
Such as polystyrene as in HPLC.
Pressure changes in SFC have a pronounced effect on
Retention and hence capacity factor. This is consequence
Of change in density with pressure. Density increases
With pressure. Increase in density lowers retnetion
As the analytes dissolve better in the mobile phase
And distribute less into the stationary phase.
The van Deemter plot for SFC indicates that for a given
Linear mobile phase velocity the HETP in SFC is about
A factor of 3 smaller than in HPLC.
When a molecule dissolves in a supercritical fluid, the
Process resembles volatilization at a much lower
Temperature than would be required for this compound
At a given temperature the vapor pressure for a large
Molecule in a supercritical fluid may be 1010
Greater than its vapor pressure in the absence of the
Fluid. As a result high molecular weight compounds
Such as biomolecules and thermally unstable compounds
Can be separated at low temperatures using
Supercritical fluid chromatography.
The analyte and the supercritical fluid must interact for
It to dissolve in the supercritical fluid. The fundamental
Thermodynamic parameter that helps to determine
If a given molecule will dissolve in a supercritical fluid
Is its fugacity.
The limitation of supercritical chromatography lies in the
Ability of the supercritical fluid to solubilize analyte
Molecules. Modifiers such as methanol and other solvents
Can be used to enhance the solubility of analytes in
Most commonly employed supercritical fluid is CO2.
The affinity of a compound for supercritical CO2 is
Termed is CO2philicity.
EFFECT OF MODIFIERS ON SOLUBILITY IN
Diuron – phenyl
Derivative of urea
TCDD – 2,3,7,8-
LAS – linear