Extraction Technologies for Medicinal and Aromatic ... - Capacity4Dev

12 FLASH CHROMATOGRAPHY AND LOW PRESSURE CHROMATOGRAPHIC TECHNIQUES FOR SEPARATION OF PHYTOMOLECULES
12.6.3 Method Development Using Gradient Elution
Method development can be performed without TLC. Gradient
elution analysis offers a useful approach to method development, particularly
for non-silica-based fl ash chromatography (e.g. reverse phase) where
suitable TLC plates are not available.
1. Load the sample onto a prewetted (equilibrated) fl ash column
in as weak a solvent as possible (or use dry loading);
for example, for normal phase work use hexane for loading.
2. Elute the column with aliquots (2 CV each) of successively
increasing solvent strength. A typical scheme for mixing the
two solvents is given in Table 5.
3. Collect each fraction and analyze for the presence of the
components of interest.
4. Using these data, identify the solvent mixture that elutes
the components of interest separately, and set up a step or
continuous gradient as described.
Table 5: Typical scheme of mixing solvents for method development.
Solvent A is a weak solvent (e.g. hexane) while solvent B is a strong solvent
(polar modifi er, e.g. isopropanol)
Aliquot number Solvent A, % Solvent B, %
1 100 0
2 99 1
3 98 2
4 97 3
5 06 4
6 95 5
7 94 6
8 93 7
9 92 8
10 91 9
12.6.4 Practical Tips for Gradient Elution
1. The use of a gradient does not improve the selectivity of a
separation if isocratic elution using the same solvent system
does not effectively separate the sample components.
However, a gradient can be used to decrease the time required
to achieve a separation.
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