Process-scale High Performance Liquid Chromatography for Medicinal and Aromatic Plants

1. Process-scale High
Performance Liquid
Chromatography for
Medicinal and Aromatic
Plants
Jedai Ejem
MS-EST 1 

2. • 1964 when the first HPLC instrument was constructed by
Csaba Horvath at Yale University.
• Preparative mode HPLC (prep-HPLC)- for isolation of
compounds; can be used in pharmaceutical development
for trouble-shooting purposes.
• The importance of prep-HPLC in pharmaceutical production
as a purification tool has been increasing.
• Chromatography can be classified according to mechanism
of separation as: adsorption chromatography, partition
chromatography, ion ex- change chromatography, size
exclusion chromatography and affinity chromatography.
• HPLC can also be classified according to special techniques,
such as reverse phase (RP) and normal phase
chromatography.

3. • Liquid chromatography has overtaken gas
chromatography, as high performance liquid
chromatography (HPLC) systems now provide
features such as:
• i) High resolving power
• ii) Fast separation
• iii) Continuous monitoring of column effluent
• iv) Qualitative and quantitative measurements and
isolation
• v) Automation of analytical procedures and data
handling

4. • HPLC played an important role in the progress of
natural product chemistry.
Theoretical Aspects of HPLC
Separation of chemical compounds is carried out by passing
the mobile phase, containing the mixture of the
components, through the stationary phase, which consists
of a column packed with solid particles.
Two Modes of Analysis
 a. Isocratic analysis- which the composition of the mobile phase
remains constant during the elution process.
 b.Gradient elution, the composition of the mobile phase changes
continuously or stepwise during the elution process.

5. • Important Factors that Influence HPLC Separation
a. Dead volume b. Capacity factor
c. Theoretical plate count d. Resolution
e. Selectivity
• Main Components of HPLC
a. Reservoir b. Degasser
c. Pumps d. Injector/auto sampler
e. Column f. Guard column
g. Detectors h. Fraction collector
I. Records

6. Advantages of HPLC
• i) Variety of separating techniques.
• ii) Variety of column packings for different techniques.
• iii) Separation optimized by alteration of the mobile phase.
• iv) Mobile phase easily manipulated in gradient systems.
• v) RP technique separates very similar and very different com-
pounds simultaneously.
• vi) HPLC can be used as a preparative method.
• vii) HPCL can be used as a purification technique. More than one
detector can be connected in series (e.g. UV and evaporative light
scattering detector).
• viii) Most sample analysis is carried out at room temperature.
• ix) Short analysis runs. More than 70% of HPLC separations are
performed on UV detectors and 15% rely on fluorescence without
any derivatization.

7. Strategy for Preparative Separation
• Selection of the appropriate mode of
chromatography
• Optimization
• Scale-up of separation
Practical Consideration in Preparative HPLC Scale-up
• Sample Loading
• Separation Time
• Solvent Composition
• Washing Steps
• Recycling

8. Problems Encountered in Preparative
Scale-up
• Purity of Crude Extract-
• Removal of Chromatographic Solvent-
• Temperature Variation from Laboratory to Pilot
Scale
• Increase in Pump Pressure Due to Accumulation of
Impurities on the Column

9. Conclusions
• Stepwise scale-up starting with analytical scale to
process scale is an important issue that needs to be
considered. Optimization of operating conditions is
always useful for getting high purity
phytomolecules. Thus, process-scale HPLC is the
choice for isolating valuable molecules with desired
purity for commercialization. Its signifi cance will
continue to grow because of the increasing
requirements for high-purity molecules.

10. Questions:
1. What is the difference between prep-HPLC to the
other chromatography?
2. What is the importance of prep-HPLC in
pharmaceutical purposes?
3. Why is it that innovation in micro-analytical to
prep-HPLC played an important role in the
progress of natural product chemistry?
4. What is the cause of retention on the
chromatographic column?
5. What happened in the impurities that
accumulates on the column during a series of
sequential runs