User manual

74 Agilent 1260 Infinity High Performance Autosampler User Manual
5 Optimizing Performance
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. This is the reason that the 1260 Infinity LC system was
designed to go to 600 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.