14
Theoretical Background
Practical Issues
2D-LC User Guide
380
Practical Issues
The table below gives an overview, which practical issues have to be considered
in 2D-LC.
Based on theory, in most cases following approaches to achieve best possible
2D-LC should be respected:
•
Methodology
As in Comprehensive 2D-LC is no direct need
1
for UV-detection in the first
dimension, other eluents than acetornitril or methanol are possible. This
implies the possibility to use unconventional organic solvents in the first
dimension.
Table 47
Practical issues in 2D-LC
Issue
Theoretical base
Comment
Choice of first
dimension column
diameter
Has impact on trade off between optimum first dimension flow
rate and amount of sample injected into the second dimension
column for each second column run
True gradient elution in the second
dimension separation provides
better peak capacity than in
isocratic elution.
Gradient elution is the best
available mechanism for achieving
peak focusing.
Ratio of column
diameter in the two
dimensions
Causes significant analyte dilution effects
Goals of the analytical
method
Chosen parameter depend on what is important in analysis:
•
separate as many constituents as possible or
•
focused on resolution and quantitation of a specific
constituent
Selection of the
stationary phases and
column formats
For RPLC in both dimensions the retentivity of the second
dimension column must be much higher than that of the first
dimension column required because:
•
a relatively large volume of the sample will be collected and
injected into the second column
•
to minimize peak broadening the sample should be focused
at the inlet of the second column
1
In case the peak and time triggered operation of the second dimension separation, which is
optionally available with the Agilent 1290 Infinity 2D-LC solution, an UV-detector is required
between the first dimension column and the modulation valve.
NOTE
Take care when using any unconventional organic solvents that these are still
compatible with the used instrumentation. In doubt, refer to the module
documentation or call Agilent.
