Principles and Practical Aspects of Preparative Liquid Chromatography

4.3.1 The concept of
focused gradients
The resolution between groups of adjacent peaks can be increased by
using a shallow gradient profile focused on a target peak. In this section
we show different focused gradient profiles and how to generate them.
Dissolving all compounds in a sample – despite different polarity and high
concentrations – is the key for robustness of the methodology. The starting
conditions in a focused gradient profile are derived from the polarity of the
target compounds. At the same time, retention has to be reached using
as low a concentration of the organic solvent B as possible. For polar
compounds good results are obtained by starting at a low percentage of
solvent B. Even the polar compounds are then retained at the column head.
After the isocratic hold, a steep gradient step can be used to ramp up
to the starting point of the focused gradient for each zone. The applied
gradient profile is shallow to achieve optimum separation efficiency for
the target compound and close eluting impurities.
For non-polar compounds the solubility is higher at larger percentages
of solvent B. The risk of precipitation and plugging of capillaries during
injection can be reduced when the starting conditions are close or even
the same as the initial conditions of the applied gradient profile for this
elution zone. After sample transfer to the column has succeeded a shallow
gradient profile will be applied around the elution zone of the compounds
of interest. After elution of these compounds the column will be purged
immediately. All other compounds are purged out. The process is optimized
to reduce the run times.
100%B
Injection
Isocratic
hold
Focused
gradient
Purge
Nonpolar
Medium polar
High polar
0%B
Time
Figure 4.4 Focused gradient profiles for target compounds with different polarities.
The dashed line represents an alternative step-gradient profile.
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