Agilent Technologies 1290 Infinity LC System System Manual And Quick Reference page 58

3
Optimization of the Agilent 1290 Infinity LC System
How to Achieve Higher Resolution
The van Deemter curve shows that the optimum flow rate through an STM
column is higher than for larger particles and is fairly flat as the flow rate
increases. Typical, close to optimum, flow rates for STM columns are:
2 ml/min for 4.6 mm i.d.; and 0.4 ml/min for 2.1 mm i.d. columns.
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
where:
• k
• t
• F = flow (ml/min),
• V
• %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
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).
Any reduction in extra-column volume will reduce dispersion and give better
resolution. This is already optimized in the 1290 Infinity LC system with
narrow bore (0.12 mm i.d.) capillaries (check that the shortest length is used
between column and detector) and the Max-light cartridge flow cell.
Finally, any gains in resolution must be preserved by having data collection
which is fast enough to accurately profile the narrow peaks.
58
*
= mean k value,
= time length of gradient (or segment of gradient) (min),
G
= column delay volume,
m
*
in the following equation:
Agilent 1290 Infinity LC System Manual and Quick Reference
*
remains constant