Towards Comprehensive Online Multidimensional Frequency Transform Separations (COMForTS) Digital Doctoral Thesis Materials – Mark J. E. Trudgett 2014
	Thesis Abstract Comprehensive multidimensional separations are today dominated by systems that are fundamentally limited to highly asymmetrical online separations sacrificing separation space, or to lengthy, time consuming offline separations. With the exception of pulse-modulated methods in gas chromatography, separations have thus been limited to two dimensions. In liquid chromatography, the highest efficiencies in terms of separation power are achieved through asymmetric comprehensive online two-dimensional separations where the capacity to separate analytes within a given time is severely restricted by the need to avoid under-sampling of the first dimension and by the subsequent limitation of peak capacity within the second dimension. These limitations have been overcome in this thesis by employing multi-dimensional detection whereby both the retention times and frequencies of analyte pulses are recorded. Within a given separation dimension, analyte pulses were related to their linear velocities. Time-dependent frequency analyses combined with knowledge of the physical dimensions of that separation dimension allowed the determination of both the times at which analytes entered that separation dimension and their retention times within that dimension. By this means, it is possible to reconstruct a virtual comprehensive multidimensional separation. This approach has been called Comprehensive Online Multidimensional Frequency Transform Separations (COMForTS). Analyte pulses can be introduced either as physical pulses resulting from alternately switched valves or as virtual pulses produced from the combination of signals from multiple in-separation detectors. The principle of operation being the same in both cases, a semi-empirical computer model of a physically pulsed system was developed and its feasibility positively demonstrated in simulations of high-efficiency separations in two dimensions. In that model, time-dependent frequency spectra were obtained by short time Fourier transforms. Performance characteristics with respect to harmonics, overtones, pulse width, peak width and peak frequencies were identified. Separations of higher dimensionality were also shown to be possible. Following this theoretical groundwork, a basic prototypical COMForTS instrument together with custom control software and signals processing system was designed and constructed based upon a less physically demanding on-column multipoint detector array. Practical online two-dimensional separations were performed in which varied and controlled degrees of peak wrap-around and physical overlap were generated, replicating the effects of first-dimension under sampling. It was simultaneously shown that while a conventional online two-dimensional separation failed, COMForTS was able to fully, and correctly, resolve all analytes. Improvements in peak production rates of between 26- and 41-fold were observed and quantitative results were obtained. Minimal interferences were observed when time-dependent frequency transforms were cross-correlated. Significant future improvements to both the efficacy and speed of signals processing were also identified. COMForTS also places no restriction on the analysis time (or peak capacity) of second, or higher separation dimensions. Total analysis times increase only additively with dimensionality whilst increases in peak production rates are multiplicative – possibly approaching three orders of magnitude in three-dimensional separations. Most significantly, COMForTS demonstrated an exceptionally high confidence in the purity of peaks, bypassing limitations imposed by current statistical peak overlap theory by applying conditional logic to the separation of analyte information. COMForTS is limited in that it provides separation information rather than a physical separation and that, in its most efficient form, detection is limited to non-destructive, arrayed, in-separation methods. Nonetheless, the method exhibits great potential for applications that demand complex separations with rapid turnaround, such as in process control, comparative metabolomics, fingerprinting of natural products and rapid screening of complex samples. It is envisaged that these applications of the method would leverage emerging high-efficiency micro- and nanoscale separations technologies facilitating close to time-of-sampling analytical results.