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5
How to Optimize the Performance of Your Module
How to Achieve Higher Resolution
length of a column and so smaller particle size and a longer column will give a
higher plate number. The pressure rises with the inverse square of the particle
size and proportionally with the length of the column. This is the reason that
the 1290 Infinity LC system was designed to go to higher pressures so that it
can run sub-two-micron particles and column length can be increased to
100 mm or 150 mm. There are even examples of 100 mm and 150 mm columns
linked to give 250 mm length. Resolution increases with the square root of N
so doubling the length of the column will increase resolution by a factor of 1.4.
What is achievable depends on the viscosity of the mobile phase as this relates
directly to the pressure. Methanol mixtures will generate more back pressure
than acetonitrile mixtures. Acetonitrile is often preferred because peak shapes
are better and narrower in addition to the lower viscosity but methanol
generally yields better selectivity (certainly for small molecules less than
about 500 Da). The viscosity can be reduced by increasing the temperature but
it should be remembered that this can change the selectivity of the separation.
Experiment will show if this leads to increase or decrease in selectivity. As
flow and pressure are increased it should be remembered that frictional
heating inside the column will increase and that can lead to slightly increased
dispersion and possibly a small selectivity change both of which could be seen
as a reduction in resolution. The latter case might be offset by reducing the
temperature of the thermostat by a few degrees and again experiment will
reveal the answer.
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
66
*
= mean k value,
= time length of gradient (or segment of gradient) (min),
G
= column delay volume,
m
*
in the following equation:
Agilent 1290 Infinity Binary Pump User Manual
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