Extraction Technologies for Medicinal and Aromatic ... - Capacity4Dev

11 PROCESS-SCALE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY FOR MEDICINAL AND AROMATIC PLANTS
Capacity factor (K ’ ), selectivity (α) and column effi ciency (N) are
three fundamental parameters that infl uence the resolution of a chromatographic
separation, as follows:
R s = 1 4
( α – 1
α
) √⎯N (
K '
1 + K ')
11.2.3 Main Components of HPLC
An HPLC system contains the following components:
a) Reservoir. This is meant for the mobile solvents. Acetonitrile, methanol,
heptane, isopropanol and cyclohexane are the organic modifi ers most
commonly used. Trifl uoroacetic acid, heptafl uorobutyric acid, phosphoric
acid and triethylamine phosphate are ion-pairing reagents for better
chromatographic results. All tubing and fi ttings should be chemically
inert. Solvent must be fi ltered through a 0.45-μm fi lter unit.
b) Degasser. In analytical operations, the mobile phase should be free of
air bubbles. For this purpose, a degasser is used.
c) Pumps. These are devices that deliver the mobile solvent at a controlled
flow rate to the separation system. HPLC uses reciprocating pumps: a
pump with a single or multiple chambers, from which the mobile phase is
displaced by reciprocating pistons or diaphragms. Binary gradients are created
by the selected mixing of two solvents, on a single-headed two-pump
system. Accurate gradient is maintained by microprocessor control.
d) Injector/autosampler. This device introduces a liquid sample into the
mobile phase or onto the chromatographic bed. An autosampler can
perform repeated functions without operator attendance, and thus is a
labor-saving device.
e) Column. Silica and modifi ed silica columns are available for various applications.
Examples are octyl (C 8 ), octadecyl (C 18 ), phenyl (C 6 H 5 ), and
cyno (CN) columns.
f) Guard column. This is used to protect the main column.
g) Detectors. No universal detector is available for all molecules. However,
according to the characteristic of the molecules investigated, various
detectors are used (Table 1).
h) Fraction collector. This device collects the fractions containing the molecules
of interest during the chromatographic run.
i) Records. A computer is used for chromatographic data acquisition.
Table 1: Characteristics of various HPLC detectors
Detector Application Advantages and limitations
Electrochemical Responds to substances that
are oxidizable or reducible
Commercially available.
Non-specifi c. High LOD
184