Extraction Technologies for Medicinal and Aromatic ... - Capacity4Dev

12 FLASH CHROMATOGRAPHY AND LOW PRESSURE CHROMATOGRAPHIC TECHNIQUES FOR SEPARATION OF PHYTOMOLECULES
2. Optimize the TLC solvent mixture so that the compound of interest
has an R f ≈ 0.15-0.35 and ΔR f >0.20. These conditions will provide
the most reliable starting point for a successful flash chromatography
separation.
a) Adjust the solvent selectivity to provide an ΔRf>0.20. Solvent selectivity is
defined as the ability to affect the retention of one compound in the mixture
relative to the others, therefore affecting ΔR f and number of CV. Experimenting
with different solvent combinations to obtain the desired TLC
separation usually reveals appropriate conditions for effective flash chromatography
separation. Different solvent mixtures such as hexane:ethyl
acetate (1:1) and hexane:dichloromethane (1:2) may provide different
solvent selectivities while providing similar solvent strengths. Different
solvent mixtures can even reverse the elution order of some of the components
in the sample. It is interesting to note that ΔR f and ΔCV may vary
greatly relative to one another for a given separation. ΔCV predicts column
capacity, i.e. the amount of material that can be effectively separated in a
single column loading (Table 2). Greater the ΔCV, the better the effective
capacity of the column.
b) Adjust the solvent strength to obtain an Rf between 0.15 and 0.35 (CV, 3-6).
Solvent strength refers to the solvent’s simultaneous effect on the retention
of all compounds in the mixture; therefore, solvent strength affects R f
and CV. Once the optimum separation has been established by modifying
solvent selectivity, it may be useful to move some or all of the compounds
off the flash column as quickly as possible by increasing solvent strength.
Often, slight changes in solvent strength can make large differences in retention.
In some cases, a lower-strength mobile phase provides improved
separations. It is important to remember that the sample loading solvent
should have equal or lower elution strength than the starting strength of
the mobile phase. Additional adjustments to the selectivity and strength
of the flash solvent system may be necessary to optimize the separation
and to achieve a CV≈3-6 and a ΔCV>1. This can often be achieved by using
a less polar solvent system or by decreasing the proportion of polar
modifier.
Table 2: Approximate capacity of a 20 g/70 ml ISOLUTE Flash Si column (Biotage)
ΔCV
Sample load, g
6 1.0
2 0.5
1 0.25
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