|Exploring the possibilities of temperature responsive columns in comprehensive two-dimensional liquid chromatography.
|Comprehensive Chromatography - The State of the Art
|Mr Mathijs Baert
|Ms Kristina Wicht
Prof Gert Desmet
Prof Andre de Villiers
Prof Frederic Lynen
Abstract Information :
The use of comprehensive two-dimensional liquid chromatography (LCxLC) can provide
the required resolving power for a satisfactory analysis of very complex samples. This
combination of two LC modes allows for a theoretical increase in peak capacity equal to the
product of the peak capacities of both modes. However, the practical implementation of this
technique is not without its own unique set of obstacles. One of the main challenges to
overcome is the problem of modulation between the 2 dimensions. When transferring the
sample from the 1st to the 2nd dimension, the volume of the fraction and the inadequate
refocusing of the sample on the 2D column will inadvertently lead to a significant loss in
peak capacity and sensitivity. Research has tried in recent years to provide maximal
suppression of these negative effects, by optimizing the modulation conditions in such way
that they minimize the lost peak capacity and sensitivity.
In previous work a novel comprehensive 2D-LC method was developed, whereby a temperature responsive separation mode (TRLC) and a fast reversed phase column (RPLC) were used in the first and in the second dimension, respectively. It was shown that the implementation of a poly(N-isopropylacrylamide) (PNIPAAm) based 1D column resulted in excellent on column refocusing of the analytes, thus providing an excellent solution to overcome the problem of modulation in 2D-LC.
In this work, we further explore the possibilities of this new comprehensive 2D-LC technique, designated as TRLCxRPLC. The inherent on-column focusing at the start of the 2D column is further exploited, with the aim to significantly improve sensitivity, by injecting larger fraction volumes and using optimized temperature responsive column dimensions. This is demonstrated though the separation of representative mixtures of solutes, whereby the influence of key aspects such as loop volume, modulation time, temperature, sample capacity, column dimensions, flow rates and resulting peak capacities are assessed to map the potential of this novel 2D-LC combination.