Agilent Technologies Agilent 1260 Infinity System User's Manual page 55

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
• 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
happens in half the time.
Any reduction in extra- column volume will reduce dispersion and give better
resolution. This is already optimized in the 1260 Infinity Binary LC 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.
In summary, the following steps should be considered to increase resolution:
• The first step in improving resolution is always to test different
• The second step is to use long columns or even coupled columns to
• A third step is to shift peaks to higher retention factors. For k values
In practice, this means that longer columns with appropriate selectivity
give better resolution.
1260 Infinity Binary LC - System User Guide
*
= mean k value,
= time length of gradient (or segment of gradient) (min),
G
= column delay volume,
m
*
remains constant and the separation looks the same but
stationary phases and to select the column with the best separation.
This is the parameter that is of most importance for resolution.
increase the plate number.
of 5 to 10, the impact is significant. With higher k values, the effect is
very low.
Optimization of the Agilent 1260 Infinity Binary LC
How to Achieve Higher Resolution
*
in the following equation:
3
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