Agilent Technologies 1260 Infinity II User Manual page 77

4
How to Optimize the Performance of Your Module
How to Achieve Higher Resolution
In isocratic separations, increasing the retention factor, k, results in better
resolution because the solute is retained longer. In gradient separations the
*
retention is described by k in the following equation:
where:
*
• k
= mean k value,
• t
= time length of gradient (or segment of gradient) (min),
G
• F = flow (ml/min),
• V
= column delay volume,
m
• Δ%B = change in fraction of solvent B during the gradient,
• S = constant (ca. 4-5 for small molecules).
This shows that k and hence resolution can be increased by having a shallower
gradient (2 to 5 %/min change is a guideline), higher flow rate and a smaller
volume column. This equation also shows how to speed up an existing gradient
*
– if the flow is doubled but the gradient time is halved, k remains constant
and the separation looks the same but happens in half the time. Recently
published research has shown how a shorter STM column (at temperatures
above 40 °C) can generate higher peak capacity than a longer STM column by
virtue of running it faster. (Refer to Petersson et al., J.Sep.Sci, 31, 2346-2357,
2008, Maximizing peak capacity and separation speed in liquid
chromatography).
1260 Infinity II Flexible Pump User Manual 77