Identification And Separation of

Optical Isomers of Medicinal Interest

Dr. Joachim N. Kinkel
Analytical Chemistry
Technical University, Georg-Simon-Ohm
Nuremberg, Germany
 Scope of the Presentation:
__________________________________
- Direct Chromatographic Separation of
Enantiomers

- The basic influencing factors and their

impact on optimization strategies

- Analytical Challenges

- Technical and Industrial Scale in

Pharmaceutical Research and Production
MMDR-5, Karachi, Pakistan, SIdentification And Separation of Optical Isomers of Medicinal Interest January, 12th., 2015, PL-2,
12.15 – 13.00 2
 Chromatography
_______________________________

Resolution Rs is governed by

Thermodynamics Retention, Selectivity

Kinetics Efficiency

The basic Equation of Chromatography

 Direct Chromatographic Separation of
Enantiomers
________________________________________

Enantioselectivity is governed by the energetic

differences of transient diastereomeric complexes,
formed with the chiral ligands and the 2 enantiomers
during their migration through the column.
 Chiral Recognition
______________________

The „Three-Point-Interaction“-Model (Dalgliesh, 1952)

 Chiral Recognition and
Thermodynamics
If the equilibrium constants are denoted by KRR and KSS, respectively,
the expression for the change in free energy,
∆G° = - RTlnK, will give the free energy difference:

∆ ∆G° = ∆G°RR - ∆G°SS, as - ∆∆G° = RTlnKRR/KSS,

where K = CSS/Cmm and KRR>KSS (arbitrary assumption).

From our definition of k‘ we know that k‘= KVs/Vm, which then

gives
-∆∆G° = RTlnk‘RR/k‘SS = RTlnα
Chiral Recognition and CSP –Design

_____________________
The scientific approach:
• The „Reciprocal Principle“ by W. Pirkle
 Chiral Recognition
______________________
Empirical alternatives (1970‘s, 1980`s) of CSP-
Design:

Chiral pool (amino acids, sugars, proteins ….)

Available synthetic reagents……
Chiral polymers…..
 The Demand for Speed:
As many enantiomer separations
per time unit !
→ How to get Enantioselectivity if there is no
universal Chiral Stationary Phase ?

- Empirical testing of a large number of

CSP/eluent combinations
- Prediction of Structure/Enantioselectivity
relationships
Automated screening of CSP‘s:
 The Demand for Speed (1990`s):
_______________________________________

Capital driven by pharmaceutical industry

demands:
- Empirical testing of a large number of CSP/eluent
combinations with automated systems (7/24)

- Prediction of Structure/Enantioselectivity relationships

Predictions based on statistical exploitation of
experimental data did not meet the expectations of
industry
 Description of Molecular Chirality and Its
Application to the Prediction of the
Chromatographic Behavior of Enantiomers on C
SP‘s
________________________________________

Computer-based approaches to property prediction in e.g.

chromatography require an adequate representation of
chirality that is able to distinguish between enantiomers.
The chirality code and Kohonen NN (2001-2006)

The problem: Binary classification as R and S (or D and

L) is efficient for labeling but not for correlating
structures with properties.

Three-dimensional atomic coordinates implicitly and

accurately represent chirality, but they cannot be used
directly as descriptors.

To solve the problem, Ruch coined the expressions

“Chirality Function & Chirality Code“
Ruch, E. Algebraic Aspects of the Chirality Phenomenon in Chemistry.
Acc. Chem. Res. 1972, 5, 49-56.
Instead of measuring chirality by a single value like Ruch,
J. Gasteiger presented a new molecular transform that
represents Chirality using a fixed set of descriptors.

Aires-de-Sousa, J.; Gasteiger, J.,

New Description of Molecular Chirality and Its Application to the
Prediction of the Preferred Enantiomer in Stereoselective Reactions.

J. Chem. Inf. Comput. Sci. 2001, 41, 369-375.

Aires-de-Sousa, J.; Gasteiger, J.
Prediction of Enantiomeric Selectivity in Chromatography. Application of
Conformation- dependent and Conformation-Independent Descriptors of
Molecular Chirality.

J. Mol.Graphics Model. 2002, 20, 373-388.

This Chirality Code includes information about the geometry of
chiral centers, properties of the atoms in their neighborhoods, and
bond lengths and distinguishes between enantiomers.

Additionally, it has been shown that such a code provides adequate

vectors for neural network input which allows the prediction of the
behaviour of enantiomers in stereoselective reactions and
chromatographic enantioseparations.

Aires-de-Sousa, J., Gasteiger, J., Gutman, I., Vidovic, D.,

Chirality Codes and Molecular Structure
J. Chem. Inf. Comput. Sci. 2004, 44, 831-836
The chirality code is a sequence of (typically 100) numbers,
being equal to the value of a certain “chirality function” at
equidistant points within a chosen interval. For molecules of
moderate size the chirality function has thousands of peaks
(maxima and minima), one for each quartet of atoms.

1. Does a sequence of 100 values of a function with

many thousands of peaks provide a faithful
representation of this function?
2. Is a chirality code a sound representation of the
structure of the underlying molecule?
 Yes

both fCDCC(x) and CDCC consist of clusters of nearlying

and partially overlapping peaks, whose

position is characteristic for a particular functional group

present in the molecule.

The following examples shown are typical:
In all the studied cases it was found that
* alcohols possess a group of peaks around x = - 90;
* amines possess two groups of peaks around x = - 40
and x = - 80;
* ethers possess a group of peaks around x = - 20;
* ketones possess a group of peaks around x = -50;
* carboxylic acids possess two groups of peaks around x = -50
(same as in ketones!) and x = - 90 (same as in alcohols!);
another weak peak is observed at x = - 160, but not in all cases.
* aliphatic compounds possess a group of peaks around x = 0
caused by C-C and C-H groups.
The results point at a rather favorable property of fCDCC(x) and
CDCC:
Their peaks can, in a transparent and chemically sound manner, be
associated with the functional groups present in the underlying
molecule. Although the conceptual and mathematical basis of the
chirality-code-approach is by no means related to any form of
molecular spectroscopy, the analogies between CDCCs and
molecular (especially IR and NMR) spectra are remarkable.
A representation of molecular chirality by a fixed-length
code was introduced, which was able to account for both
central chirality and axial chirality.
Its chemical significance was demonstrated by two
applications in chiral chromatography.

For a group of 28 racemates the neuronal net was able to

predict elution order, range of retention and selectivity on
a teicoplan-type CSP with a success rate > 80%.
The situation in a pharmaceutical research
laboratory (1995 – 1998):
_____________________________________
Good points:

CSP‘s based on Cellulose/amylosederivatives adsorbed

to Silica proved to be broadly applicable (95% of all
separations could be performed on 5 different packings)
(analytical scale)

These packings showed high laoading capacity at

favourable eluent condition
( preparative separations , technical scale)
The situation in a pharmaceutical research
laboratory (1995 – 1998):
_____________________________________
Bad points:

-their kinetic mass tranfer properties and their limited

chemical stability were not ideal ,

- the high purchasing costs of packing

- uncertainties due to availability and long term useage in

preparative applications (emerging SMB technology)
were far away to meet the demand of managers in
pharmaceutical companies.
The situation in a pharmaceutical research
laboratory (1995 – 1998):
_____________________________________
New mass sensitive detectors in Liquid Chromatography
LC-MS helped to trigger another methodology known
for a long time to some specialists in the oil industry:

Supercritical Fluid Chromatography (SFC)

The application of SFC-devices with packed CSP colums

under non-SFC conditions (= subcritical conditions)
helped to improve efficiency, separation speed and
reduced the number of trial-and-error experiments
 SFC -
Supercritical Fluid
Chromatography
An Alternative Separation
Method
 SFC - Supercritical Fluid
Chromatography
• An Alternative Separation Method
• High efficiency
• Improved economy
• Green process
 What is SFC?
 Supercritical Fluid Chromatography
 Uses the specific physical properties of supercritical solvents
 Higher efficiency for chromatography with capillary and packed columns
 Advanced technical requirements in systems and peripheral installations
 Typical solvent: carbon dioxide
 Cheap
 Availability; nearly unlimited (waste from industry )high purity)
 Environmentally friendly (“green chemistry”)
 Rather safe (non-toxic, but suffocating!)
 Alternatives
 Propane, trifluoro-methane, xenon, ammonia,…
Disadvantages
 Advanced requirements of technical equipment
 Modified CO2 Pump (cooled pump head)
 Modifier pump
 Column oven
 Backpressure regulator
 Pressure-resistant detector flow cell (400 bar)
 Controlled decompression of fractions (cyclone)
 Gas supply
 Cylinders with liquid CO2 and diving tube, 35 or 50 kg, bundle of 6 or 12 cylinders
 Tanks of 1 to 5 cubic meters (requires a booster pump)
 Users anxieties and beliefs:
 Complicated and dangerous technology(?)
 Unreliable, questionable robustness and reproducibility
 New technology, complexity of control software
 (Re-) validation
Supercritical fluids – phase diagram CO2

05/25/20 31
20
CO2 - transition
Supercritical Fluid Chromatography
________________________________________

In 2000, the majority of experts in preparative

chromatography did not predict a bright future
for SFC equipment.

In 2015, leading analytic labs of major

pharmaceutical companies perform > 80% of all
RP separations with SFC instrumentation.
 Supercritical Fluid Chromatography:
Operational Conditions
________________________________________
It is time to clarify,

that in the majority of cases the use of CO2 in

combination with low-percentage polar modifiers
(alcohols, acetonitrile, ethers etc.), isocratic or
gradient mode, is performed at

subcritical conditions.

The benefit of improved diffusivities is preserved!

 Supercritical Fluid Chromatography:
Équipment
________________________________________
The use of CO2 in combination with low-
percentage polar modifiers (alcohols, acetonitrile,
ethers etc.), isocratic or gradient mode,
does not require to buy new equipment!

Every HPLC unit can be easily adopted by some

minor modifications :
a. Eluent supply (liquid CO2 bottle)
b. Pump head thermostatisation (cooling, peltier)
c. Pressure stable detector cell and back pressure
regulator (set of restriction capillaries)
Supercritical Fluid Chromatography:
Equipment
Home-made pumphead for preparative SFC (2015)
Dr. Eric Francotte, Novartis Pharma,
Switzerland
 The situation in a
pharmaceutical research laboratory (2015):
_____________________________________
The need to improve the speed of analysis further
increases!

Strategy: apply generic conditions and widen the

range of modifiers.

UHPLC/SFC combinations are recommended.

Where are the drawbacks?
 The situation in a
pharmaceutical research laboratory (2015):
_____________________________________
Structures of immobilized Polysaccaride-derived
CSP‘s:
 The situation in a
pharmaceutical research laboratory (2015):
_____________________________________
 The situation in a
pharmaceutical research laboratory (2015):
_____________________________________

Elution conditions: 5 -50% modifier; 6,5%/min., total cycle time: 12,4 min, 4 ml/min,
120 bar, column dimensions: 250x4,6mm,
 The situation in a
pharmaceutical research laboratory (2015):
_____________________________________
 The situation in a
pharmaceutical research laboratory (2015):
_____________________________________
 Preparative enantiomer separation:
Research laboratory (1990)
__________________________________
 Preparative enantiomer separation:
Research laboratory (1995)
__________________________________
 Preparative enantiomer separation:
First production-scale SMB (1995)
__________________________________
NOVASEP
Varicol 5 - 1200 (2015)
 Preparative enantiomer separation:
Research laboratory (2015)
__________________________________

0,1g
racemate
Cycle time:
4,0 min

0,8g
racemate
Cycle time:
4,0 min

Specific productivity:
185g product/kg packing/day @
30mg racemate/mL MeOH
Preparative SFC 2015

Supersep 50 Supersep 300

Summary

The direct chromatographic enantiomer

separation has reached a mature situation.

Whatever dimension ,

- analytical, -technical or industrial scale-

fast, economical and green technologies are

available and ready to be used.

New control systems will give flexibility and

Improved security
Acknowledgements

H.-G. Döteberg,
H-D Separation GmbH,
Aachen, Germany

Pilar Franco, Dieter Heckmann,

Chiral Technologies,
Strassbourg, France

Eric Valery, R.-M.. Nicoud,

Novasep, Nancy, France