Extraction Technologies for Medicinal and Aromatic ... - Capacity4Dev

14 QUALITY CONTROL OF MEDICINAL AND AROMATIC PLANTS AND THEIR EXTRACTED PRODUCTS
BY HPLC AND HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY
14.5.3 Columns
Columns come in varied sizes, structural architecture and
chemistry. The chromatographic material is usually packed in stainless steel
casing. The material is composed of porous particles which vary in nature
(inorganic ceramic, organic polymer, or hybrid), shape (irregular or spherical),
size (ranging from 2 to 20 μm; normally around 5 μm) and surface
modifi cations (silanes of different carbon lengths, aminopropyl, diol, cyano,
sulphonic acid and ammonium ions). The choice of column is based on the
type of analysis. Comprehensive information is available on the websites of
the leading manufacturers of HPLC columns, which serves as good guides
in choosing columns for analysis. Most analyses are reported on reverse
phase columns, usually C 18 , with increasing emphasis on reducing the column
length, diameter and analysis time. Most HPLC separations are successful
on columns maintained at ambient temperature, but thermostatted
column maintained to ±0.2° C is necessary for reproducible results. This is
because all mechanisms of separation are temperature-dependent and any
shift in temperature has remarkable bearings on the result.
14.5.4 Detectors
A wide variety of detectors is available to cater to diverse needs
of the analysts. Ultraviolet detectors of fi xed wavelength, dual wavelength or
variable wavelength (photodiode array detector) are most frequently used.
Other options are refractive index detector, fl uorescent detector, electrochemical
detector, evaporative light scattering detector and chemiluminescence
detector.
14.5.5 Data Processing
The electrical response from the detector is digitalized and fed
to a data processing module, which in present days is invariably a computer,
and computations are made using special software. Several software programs
are available for data processing, from both manufacturers and third
parties. Besides computing the data, they also control the entire operation
of the machine.
14.5.6 Factors Affecting HPLC Analysis
Numerous variables affect an HPLC analysis. This topic is beyond
the scope of this paper, but some critical variables are discussed briefly.
Increased emphasis is now paid to control the temperature of the column
within a narrow range to ensure precision of the result. This is desirable, as
factors such as solubility, solute diffusion, viscosity of the mobile phase, and
column plate number all are affected by temperature. Mobile phase composition
is another vital parameter that affects the resolution, retention time
and peak area. Pumps contribute the most towards variations of results, as
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