Comprehensive two-dimensional liquid chromatography (LC×LC) offers some very attractive features. It allows the separation of very complex, non-volatile mixtures in a relatively short time and it yields structured, readily interpretable chromatograms if the separation dimensions (i.e. the retention mechanisms) match the dimensions (i.e. molecular structure) of the sample. LC×LC separations may indeed be “comprehensive” in the sense that they may reveal information that is unattainable or overlooked using modern LC-MS techniques.
To make full use of the potential of LC×LC it is tantamount that completely different (“orthogonal”) separation mechanisms are employed in the two underlying dimensions. Orthogonal separations can rigorously be realized in some situations, notably for the separation of synthetic polymers based on two clearly defined sample dimensions (e.g. molecular weight, polymer composition, end groups, branching). In the more general context of separating complex mixtures with many different sample dimensions (many different molecular structures) truly orthogonal separations may not easily be achieved. A combination of RPLC and HILIC is principally attractive, but has limited applicability, whereas combining RPLC and NPLC is practically challenging.
To overcome incompatibility problems associated with the use of first-dimension eluents as second-dimension injection solvents and to avoid injection band broadening in the second dimension the development of “active” modulators is essential. This implies that the analyte solution collected from the first dimension is focused and concentrated before being injected in the second dimension.
In this lecture we will discuss orthogonal separations, possible modulation principles and the systematic development of LC×LC separations.