The analysis of very complex sample mixtures constitutes a major analytical challenge and generally requires a high-resolution chromatographic separation prior to the detection of individual sample constituents for identification and/or quantification purposes. An example of an application area where sample complexity may hamper the analysis is proteomics research, e.g., biomarker discovery.
Current quantitative proteomics approaches rely on liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) and typically allow to routinely assess more than 5000 protein groups and in some cases even more than 10,000 protein groups. However, even with the introduction of ultra-high-performance liquid chromatography and using longer (up to 1 m) columns packed with sub-2-micron stationary-phase particles the resolving power remains insufficient to tackle contemporary life-sciences sample mixtures. Moreover, this compromises throughput. Two-dimensional liquid chromatography (2D-LC) allows to significantly increase the peak capacity, although not up to a level that is required in modern proteomics.
Spatial comprehensive three-dimensional chromatography (3D-LC) offers an innovative approach to achieve unprecedented resolving power in terms of peak capacity and sample throughput. This advanced technique separates components within a 3D separation space, where orthogonal retention mechanisms are incorporated. The parallel development of the second- and third-dimension stages effectively overcomes the inherent limitation of conventional multidimensional approaches, where sampled fractions are analysed sequentially, while high separation power is realized given that the maximum peak capacity is the product of the three individual peak capacities.
During this presentation, the intricate design aspects and prototyping of integrated microfluidic chips for spatial 3D-LC will be discussed. The discussion will extend to exploring novel engineering solutions imperative for achieving precise flow confinement throughout the successive 1D, 2D, and 3D development stages. Additionally, an innovative robotic approach for interfacing with mass spectrometry detection will be presented. Finally, the separation performance, in terms of peak capacity and peak-production rate will be put in perspective with conventional 1D-LC and two-dimensional LC approaches.
