Extraction Technologies for Medicinal and Aromatic ... - Capacity4Dev

EXTRACTION TECHNOLOGIES FOR MEDICINAL AND AROMATIC PLANTS
As a general guideline, the amount of sample loaded onto the
normal phase fl ash column should be 5%-10% of the column size. Factors
affecting the capacity of the column include compounds, sample matrix,
concentration of reaction products and elution solvent used.
12.6 Elution
12.6.1 Step Gradient Elution
Step gradients provide controlled elution with discrete changes
in eluent strength. Each step is optimized to elute only those components
that are soluble in that eluent. This technique can be applied to both off-line
and on-line fl ash chromatography.
1. Use TLC to determine a suitable solvent strength to elute
the components at discrete intervals, choosing different solvent
mixtures that elute each component separately with an
R f of 0.2-0.5.
2. Calculate the volume of solvent required to elute each component
using CV=1/R f . Values of CV for different column sizes
are listed in Table 3.
3. Apply between 2 and 5 CV of solvent for each step, starting
with the solvent with weakest strength.
4. Collect the eluent from each step in a separate vessel.
12.6.2 Linear Gradient Elution
Liner gradients are a quick way of separating complex mixture,
reducing the complexity of the subsequent purifi cation of the fractions collected.
This technique is suitable for on-line fl ash chromatography.
1. Use TLC to fi nd both the weakest and strongest elution
solvent. The weak solvent (solvent A) should give retention
of the majority of components (R f 0.5).
2. Run a gradient starting with 100% solvent A and ending with
100% solvent B.
3. Collect the eluent at regular intervals.
Automated fl ash chromatography instrumentation such as the
FlashMaster II and Solo systems can monitor the signal from a UV detector,
and collect only the fractions of eluent carrying the compounds of interest.
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