The present proposal assembles a team of separation scientists, analytical chemists and 2 computer scientists to further develop high speed two-dimensional liquid chromatography 3 (2DLC). Our principal goal is to transform 2DLC from a niche technique for use on complex 4 samples with 1000s of components into a more practical, accessible method. We will make and 5 characterize a number of innovative stable stationary phases, including a weak acid cation 6 exchanger that we believe will be ideally suited for mass spec analysis as well as novel, 7 zwitterionic and very highly hydrophilic phases designed for use as RPLC and HILIC phases for 8 2DLC. After characterization, we will focus on high-speed, robust application of these materials 9 in biomedical studies and the analysis of pharmaceuticals and environmental and workplace 10 toxicants. Stable phases are critical for use as the ultra-fast second dimension column when we 11 use high temperature LC to implement high speed 2DLC. We now generate effective (not 12 hypothetical) peak capacities of well over 1,000 in a total time of 30 minutes, thereby reaching 13 rates of two peaks/second. In prior work, 2DLC was applied to a tryptic digest (IEX x RPLC), the 14 low molecular weight components of native and mutant maize extracts, as well as urine, wine and 15 coffee samples, all using RPLC x RPLC. More important, the limitations of 2DLC were studied 16 and a systematic comparison to fully optimized 1DLC was initiated. We showed that 2DLC 17 produces more resolution than fully optimized 1DLC in times of only about 10 minutes. Our 18 work shows that it will be important to increase the speed of the second dimension even further. 19 Thus, by additional improvements in instrument and column design, we propose to increase the 20 speed of the second dimension without compromising peak capacity. Moreover, we will develop 21 a novel mode of separation-""""""""dynamic gradient elution""""""""-to maximize use of the 2D separation 22 space and thus peak capacity. We will explore the potential of unusual RPLC solvents and a 23 novel stationary phase for optimizing the first separation dimension. We propose the exploration 24 of chemically selective pre-fractionation methods to create a """"""""pseudo"""""""" three-dimensional 25 separation. Initial efforts will focus on chemically selective methods of carbohydrate separation 26 and their application to glycomics. A series of theoretical studies aimed at understanding the 27 compromises involved in optimizing 2DLC separations will also be done. Finally, retention time 28 alignment in 2DLC is much more difficult and more serious than in 2DGC. We will approach this 29 problem by several new chemometric routes so that long-term metabolomic studies can be carried 30 out by 2DLC, which is clearly superior to 2DGC for biological materials.
The vast majority of all biological, medical, agricultural and environmental experiments and measurements involve the analysis of extremely complex mixtures containing hundreds, if not thousands, of different chemical entities spanning a million fold or greater range in relative amounts. The analysis of these mixtures only becomes possible when the chemicals of interest can be separated from all interfering substances. The object of this work is to improve the separating power, and especially the speed, of liquid chromatography to allow the detailed, high resolution study of complex biological mixtures and thus make it possible to find the biomarkers of disease as well as promote our understanding of the metabolism of foods and pharmaceuticals.
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