Agilent Technologies Infinity II 1290 User Manual page 44

4
How to Optimize the Performance of Your Module
How to Achieve Higher Resolution
ways. N is inversely proportional to the particle size and directly
proportional to the 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 1200 bar 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
44
*
= mean k value,
= time length of gradient (or segment of gradient) (min),
G
*
in the following equation:
Agilent 1290 Infinity II Flexible Pump User Manual