Extraction Technologies for Medicinal and Aromatic ... - Capacity4Dev

EXTRACTION TECHNOLOGIES FOR MEDICINAL AND AROMATIC PLANTS
mixture, a column to effect separation, a detector to reveal the presence of
analyte in the eluate, and a suitable data processing unit.
14.5.1 Pumps
The pump is considered a heart of the HPLC system, as all
depends on the composition of the mobile phase and its fl ow rate accuracy.
The pump gives a pulse-free fl ow of mobile phase; the expected variations
in fl ow rate are less than 1.0%. Online mixing of solvents is preferred to
manual mixing. However, compositions containing less than 10% of a particular
solvent are better prepared by manual mixing. The composition of the
mobile phase is either constant during the analysis (isocratic mode) or it is
changed (gradient mode). The type and design of modern pumps allow low
pressure mixing of up to four solvents; else, different pumps, one for each
solvent, are required for gradient operation and the solvents are mixed at
high pressure. A typical analytical procedure uses a fl ow rate of about 1 ml/
min and operating pressure between 1000 and 2000 psi. Higher fl ow rates
generating higher pressure should always be used with justifi cation, as they
decrease column life besides requiring frequent servicing of the pump.
The fl ow accuracy of the pumps is critical for analysis. The constancy
of retention time of the last eluted peak is a measure of long-term
fl ow accuracy of the pumps, whereas short-term fl ow accuracy is checked
from the average peak areas of each component and their standard deviations.
The mobile phase must be free of dissolved gases to ensure an accurate
fl ow and to minimize noise due to bubbles. Vacuum fi ltration, sonication
and helium gas purging are methods for degassing.
14.5.2 Injector
The injector allows a predetermined volume of test solution to
be introduced into the fl ow channel of the system, without disturbing the
fl ow kinetics. Typically, fi xed volume injections are preferred over variable
volume injections. When using fi xed volume loops, it is advisable to fl ush
higher volumes of the sample through the loop to ensure complete fi lling of
the loop with the sample solution. The mobile phase close to the inner walls
of the loop can only be assured to have pushed out after injecting volumes
larger than the loop volume, e.g. injecting 20 μl test solution into a 20-μl
loop cannot assure accurate injection; if the quantity of the test material
is not a problem, fl ush the loop with over 100 μl test solution. Only the appropriate
needle (compatible with the injection port) should be employed
for the purpose of making an injection. It is important to select a syringe of
appropriate size when giving variable injections; a thumb rule for any analytical
technique is not to use the volumetric apparatus if less than 20% of its
total volume is being used. Thus, a syringe of 25 μl should not be used to
measure or inject volumes less than 5 μl.
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