Agilent Technologies 1260 Infinity II User Manual page 76

4
How to Optimize the Performance of Your Module
How to Achieve Higher Resolution
The resolution equation shows that the next most significant term is the plate
count or efficiency, N, and this can be optimized in a number of 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. 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.
76 1260 Infinity II Flexible Pump User Manual