Extraction Technologies for Medicinal and Aromatic ... - Capacity4Dev

11 PROCESS-SCALE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY FOR MEDICINAL AND AROMATIC PLANTS
form of automation: thus, developing well-automated preparative chromatographic
methods is a necessary but demanding task.
Innovations in micro-analytical to preparative HPLC played an
important role in the progress of natural product chemistry. HPLC is used
routinely in phytochemistry to pilot the preparative-scale isolation of natural
products and to control the fi nal purity of the isolated compounds. The
development of hyphenated techniques related to this effi cient separation
technique in the past 20 years has provided powerful new tools such as
LC/UV-photodiode array detection, LC/mass spectrometry (LC/MS) and LC/
NMR. The combination of high separation effi ciency of HPLC with these different
detectors has made possible the acquisition of data on an LC peak
of interest within a complex mixture.
11.2 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.
The cause for retention is physical and chemical forces acting between the
solute and the two phases, on the chromatographic column. The reason
for retention is the difference in the magnitude of forces; this results in the
resolution and hence separation of the individual solutes. The separation of
compounds occurs by distribution of solutes between the two phases.
11.2.1 Chromatography Classification
Chromatography can be classifi ed according to mechanism of
separation as: adsorption chromatography, partition chromatography, ion exchange
chromatography, size exclusion chromatography and affi nity chromatography.
In HPLC, separation is mainly governed by adsorption and partition
chromatography. In adsorption chromatography, separation is based on the
difference between the adsorption affi nities of the sample components on
the surface of an active site, whereas in partition chromatography separation
is mainly based on the difference between the solubility of sample
components in the stationary phase and the mobile phase.
There are two modes of analysis depending on the operation
techniques viz. isocratic and gradient. Isocratic analysis is the procedure
in which the composition of the mobile phase remains constant during the
elution process. In gradient elution, the composition of the mobile phase
changes continuously or stepwise during the elution process. HPLC can also
be classifi ed according to special techniques, such as reverse phase (RP)
and normal phase chromatography. Reverse phase is an elution procedure
used in liquid chromatography where the mobile phase is signifi cantly more
polar than the stationary phase. On the other hand, in the normal phase
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