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Optimization of the Agilent 1290 Infinity LC System
How to Achieve Higher Resolution
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the decision on phases it is likely that short columns were used for fast
analysis in each step of the scouting.
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 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.
Agilent 1290 Infinity LC System Manual and Quick Reference
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