Advantages of HPLC with Ultra Stable Phases

描述（由申请人提供）：本提案组建了一个由分离科学家，分析化学家和2名计算机科学家组成的团队，以进一步发展高速二维液相色谱（2DLC）。我们的主要目标是将2DLC从一种利基技术转化为一种更实用、更容易获得的方法，这种技术可用于具有1000个组件的复杂样品。我们将制造和表征一些创新的稳定固定相，包括我们认为非常适合质谱分析的弱酸性阳离子交换剂，以及设计用于RPLC和HILIC相的新型两性离子和非常高亲水性相。在表征之后，我们将专注于这些材料在生物医学研究和药物、环境和工作场所毒物分析中的高速、稳健应用。当我们使用高温LC实现高速2DLC时，稳定相作为超快速二维色谱柱是至关重要的。现在，我们在30分钟的总时间内生成了远远超过1,000个的有效（而不是假设的12个）峰值容量，从而达到每秒两个峰值的13个速率。在之前的工作中，2DLC应用于色氨酸消化（IEX x RPLC），原生和突变玉米提取物的14种低分子量成分，以及尿液，葡萄酒和15种咖啡样品，均使用RPLC x RPLC。更重要的是，研究了2DLC的局限性16，并与完全优化的1DLC进行了系统比较。我们发现2DLC 17比完全优化的1DLC在大约10分钟内产生更高的分辨率。我们的工作表明，进一步提高第二次元的速度是很重要的。因此，通过对仪器和色谱柱设计的进一步改进，我们建议在不影响峰值容量的情况下提高第二次元的速度。此外，我们将开发一种新的分离模式——“动态梯度洗脱”——以最大限度地利用二维分离空间，从而达到峰值容量。我们将探索不寻常的RPLC溶剂和23新型固定相的潜力，以优化第一分离维度。我们建议探索化学选择性预分馏方法，以创建“伪”三维分离。最初的努力将集中在碳水化合物分离的化学选择性方法及其在糖组学中的应用。还将进行一系列理论研究，旨在了解优化2DLC分离所涉及的27个妥协。最后，2DLC中的留存时间调整比2DGC更加困难和严重。我们将通过几种新的化学计量学途径来解决这个问题，这样2DLC就可以进行长期的代谢组学研究，这显然优于生物材料的2DGC。公共卫生相关性：绝大多数生物、医学、农业和环境实验和测量都涉及对含有数百种（如果不是数千种的话）不同化学实体的极其复杂的混合物的分析，这些化学实体的相对量跨越100万倍或更大的范围。只有当感兴趣的化学物质能从所有干扰物质中分离出来时，才有可能对这些混合物进行分析。这项工作的目的是提高液相色谱的分离能力，特别是速度，以便对复杂的生物混合物进行详细、高分辨率的研究，从而使发现疾病的生物标志物成为可能，并促进我们对食物和药物代谢的理解。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.