5 How to Optimize the Performance of Your Module
How to Achieve Higher Resolution
1260 Infinity II Flexible Pump User Manual 96
How to Achieve Higher Resolution
Increased resolution in a separation will improve the qualitative and quantitative
data analysis, allow more peaks to be separated or offer further scope for
speeding up the separation. This section explains how resolution can be
increased by examining the following points:
• Optimizing selectivity
• Smaller particle-size packing
• Longer Columns
• Shallower gradients, faster flow
Resolution between two peaks is described by the resolution equation:
where
• R
s
=resolution,
• N=plate count (measure of column efficiency),
• =selectivity (between two peaks),
• k
2
=retention factor of second peak (formerly called capacity factor).
The term that has the most significant effect on resolution is the selectivity, ,
and practically varying this term involves changing the type of stationary phase
(C18, C8, phenyl, nitrile etc.), the mobile phase and temperature to maximize the
selectivity differences between the solutes to be separated. This is a substantial
piece of work which is best done with an automated method development
system which allows a wide range of conditions on different columns and mobile
phases to be assessed in an ordered scouting protocol. This section considers
how to get higher resolution with any chosen stationary and mobile phases. If an
automated method development system was used in 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. Resolution increases with the
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