2D-LC User Guide 369
14 Theoretical Background
Theoretical basis of 2D-LC
General
Definition
Alternatively peak capacity may be defined as the ratio of the total area A of the
chromatogram to the area A
0
required for the resolution of any zone:
n
c
defined that way is related to the geometrical definition by a factor:
Limits of Peak
Capacity in
2D-LC
Under ideal circumstances (orthogonality), the overall peak capacity (n
c,2D
)
should be equal to the product of the individual peak capacities of the first and
second dimension separations (
1
n
c
and
2
n
c
)
In practice the increase in peak capacity is not directly proportional to increase in
ability to resolve peaks.
Probable reason for this:
• In 1D-LC, with a baseline width of a single component peak x
0
= 6, x
0
units of
component free space on both sides of the maxima is necessary to ensure
baseline resolved peaks.
• In 2D-LC the single component zone is A
0
= 2r
2
and an area of component
free space of (2r)
2
.
• As a result: For every two component free widths in one dimension, four
component free areas are required in two dimensions.
Conclusions
for 2D-LC
1D-LC is inadequate for the separation of complex mixtures, as the number of
observable peaks compared to number of peaks to observe is too low. One
theoretical model (Statistical Model of Overlap = SMO), that correlates well with
real world observations, predicts, that the maximal fraction of the total peak
capacity that can be seen as chromatographic peaks is 37 % and even only 18 %
as single peaks. This implicates that extremely high peak capacities are needed
to separate complex samples with lots of components which is extremely
difficult to achieve.
Compared to 1D-LC separations, it's complicated to predict the number of
observable peaks in 2D-LC. For example, at a given peak capacity and a given
number of components, the aspect ratio in the two axes of separation has
impact on how effective the two separation are.
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