Two-dimensional liquid chromatography has received increasing interest due to the rise in
demand for analysis of complex chemical mixtures. Separation of complex mixtures is hard to
achieve as a simple consequence of the sheer number of analytes, as these samples might contain
hundreds or even thousands of compounds. As the column-technology and pressure capabilities
in (U)HPLC has been pushed to the limit, attention has shifted towards further development of
the basic concept of HPLC - with multi-dimensional separation receiving a significant interest
within the last few years.
The focus of this thesis is on online comprehensive two-dimensional liquid chromatography
(online LC×LC) with reverse phase in both dimensions (online RP×RP).
Since online RP×RP has not been attempted before within this research group, a significant part
of this thesis consists of knowledge and experience gained in the basics of online RP×RP. The
theory behind online LC×LC is presented and discussed, and several online LC×LC systems
have been built and are described as part of the thesis. These systems were made from separate
HPLC systems not designed for online LC×LC. The lessons learned while building these
systems are described and related to online LC×LC theory. The overall conclusion on the
building of online LC×LC systems is that using standard HPLC systems for building online
LC×LC systems should be discouraged, since commercial instruments have become available.
These commercial instruments do not have limitations in inter-instrumental communication
(section 4.4), software design (section 4.5), and additional extra volumes (section 4.6), that a
home-built system is going to have, as a consequence of standard HPLC equipments not having
been designed for online LC×LC. If the choice is made for a home-built system, higher grade
HPLC systems are preferable – especially in the second dimension.
The choice of column combination in online RP×RP is an important one, and previous methods
for column selections have been focused on choosing columns with different selectivity for a
wide range of compounds. In this thesis (chapter 5), and in manuscript A, a method is presented
for a more targeted approach to choosing column combinations. This approach uses the
properties of the analytes to scale the importance of the selectivity properties of the columns.
This method significantly increases the effective use of the two-dimensional separation space.
Optimization of gradients in online RP×RP is more difficult than in normal HPLC as a result of
the increased number of parameters and their influence on each other. Modeling the coverage of
the compounds across the two-dimensional chromatogram as a result of a change in gradients
could be used for optimization purposes, and reduce the time spend on optimization. In this
thesis (chapter 6), and manuscript B, a measure of the coverage of the compounds in the twodimensional
separation space is defined. It is then shown that this measure can be modeled for
changes in the gradient in both dimensions.
As a consequence of the conclusions made within this thesis, the research group has, for the time
being, decided against further development of online LC×LC systems, since it was not deemed
ideal for the intended application, the analysis of the polar fraction of oil. Trap-and-elute multidimensional
systems are being developed for this application instead.