Molecular Approaches to Chromatography Using Single Molecule Spectroscopy

Chromatography is an important analytical technique for the separation of molecules in environmental, pharmaceutical, medicinal, natural product synthesis research, and industrial production. Despite chromatography’s extensive use, the selection of appropriate column conditions is driven by empirical methods and phenomenological theories. Single molecule spectroscopy (SMS) offers the possibility to extract molecular-scale data, with the overall goals of obtaining a mechanistic understanding of chromatography and providing a framework for intelligent chromatographic optimization, neither of which is achievable through traditional ensemble-averaged methods. Here we review both the spectroscopic techniques and the new insights that SMS has provided on interfacial liquid chromatographic separations. The experimental studies include reverse phase, normal phase (silica based), and ion-exchange chromatography. We discuss how single molecule results can inform theory and predict column performance and a perspective of future directions in the field is given. Overall, this review demonstrates the value of collaborations between the separations and single molecule spectroscopy communities and hopefully will inspire future efforts to achieve a molecular-scale understanding of the crucial analytical technique of chromatography. Chromatographic separation of molecules from complex mixtures is an important analytical technique. In the pharmaceutical industry, chromatography is used to isolate therapeutic biomolecules produced by recombinant-engineered bacteria for safe products to be consumed by patients.1 Similarly, in the natural food product industry, chromatography can quantify the amount of antioxidants or beneficial lipid products in fortified food used for maintaining health.2 Chromatographic methods are crucial in these two industries that combined accounted for over $120 billion dollars to the economy in 2009.3,4 Chromatography also has important roles in the oil and gas industry,5 environmental analysis,6 and natural product synthesis. Thus, as one of the most commonly used analysis techniques spanning many applications,7 understanding and improving chromatography has important scientific and economic implications. Recent advancements in chromatography address the needs of these diverse applications. Liquid chromatography column stationary phases improved by decreasing the particle size to <2 μm,8 using solid core-porous shell particle geometries,9 utilizing slip-flow properties of the mobile phase along the stationary phase column walls,10−12 combining hydrophilic bonded phases and ionic ligands for mixed-mode capabilities,13 and using monolithic materials14 to decrease data acquisition times, pressure requirements, and column lengths.7,15 In improving data analysis, multidimensional methods improved quantification of analytes from nonuniform peaks due to background contributions, retention time shifts, and peak shape changes.16 Theoretically, numerical and molecular mechanical modeling of analyte adsorption are used to understand plate- and mass-transfer descriptions of column performance.17,18 Despite the importance of and advancements in chromatography, an experimental molecular-scale understanding is lacking. In industry, selection of appropriate mobile and stationary phase conditions is often empirically determined through a time-intensive, costly process of testing numerous combinations of variables such as stationary phase packing density, ligand loading, and particle size; mobile phase ionic strength, hydrophobicity, and pH; and column length, diameter, and flow rate. Current explanations of chromatographic performance through theoretical models have relied heavily on phenomenological descriptions that use either variables that have no clear physical parallel within the experiment or broad definitions of diffusion, packing, and kinetics comprised of many complicated molecular processes contributing in sum. One cause of the lack of mechanistic information in both chromatographic experiment and theory is ensemble averaging. The ensemble averaged information obtained from classical analysis of a vast number of molecules inherently averages out any underlying analyte and/or process heterogeneity.19 Ensemble methods therefore make it difficult to resolve a fundamental, molecular viewpoint of the potentially heterogeneous processes that occur in practical chromatographic separations. SMS is a technique that can fill this gap. By observing one molecule at a time, heterogeneity that is hidden in ensemble-averaged studies can be revealed. For example, non-Gaussian peaks due to fronting or tailing are a challenge in chromatography (Figure ​(Figure1,1, solid line) and arise from multiple sub-populations of dynamic interactions between the analyte and stationary phase. SMS can resolve individual events that correlate and distinguish the subpopulations (Figure ​(Figure1,1, dashed lines), revealing the causes of peak broadening and asymmetry in chromatography from a mechanistic perspective not possible through traditional techniques. Therefore, SMS represents a promising path to a genuinely predictive, molecular understanding of the chromatography processes. Figure 1 Illustration of asymmetric chromatography peak that at the ensemble level (solid line) cannot resolve the heterogeneous, multiple populations present (dashed lines) that SMS can reveal. We will review the recent work in relevant SMS techniques with applications to chromatography. Work from the first reports on SMS chromatography in 1998 to the present are included but recent advancements from 2014 that use super-resolution imaging, particle tracking, and relating experimental results to theory will be highlighted. First, the underlying principles of the applicable SMS instrumentation will be summarized. Next, we will highlight specific examples of the application of SMS in providing mechanistic insight into separation methods. Throughout, we will discuss techniques and problems that have been addressed and identify scientific questions that still remain for future collaborations between the separations and SMS fields.