The study of the details of mass transfer in liquid chromatography is of central interest. We apply the microscopic (or molecular dynamic) model of chromatography to study the reversed phase separation of small and large molecules. The microscopic theory of chromatography describes the evolution of a chromatographic peak as the random migration of the molecules along the column combined with adsorption–desorption processes that occur at random, too.
The molecular dynamic model is rather straightforward to comprehend and it can furnish direct answers when one tries to understand the development of chromatographic peaks. We show that the microscopic model can be rather simply used to estimate the fundamental characteristics of the separation process. We can estimate the rate a molecule is adsorbed on the surface of the stationary phase while it migrates along the column.
When combining the general rate model with the molecular dynamic model, one can consider and compare the kinetics of the transfer of solute molecules between the flowing and stagnant zones of mobile phase, the pore diffusion, etc. We analyze the peak shapes recorded under linear conditions, and can characterize the heterogeneity of the surface of the stationary phase. With a peak shape analysis that is based on the molecular dynamic model of chromatography, we can identify the presence of heterogeneous mass transfer or adsorption kinetics. We can, furthermore calculate the amount of retention due to the individual adsorption sites. The general rate model of chromatography, which is a macroscopic model, offers the most detailed description of the separation process. We compare the results provided by both microscopic and macroscopic analysis of the peak shapes and statistical moments. In this paper we discuss the influence of wide pore-size distributions. We present results obtained on nonporous, fully porous, and fused-core particles. Furthermore, mass transfer in supercritical fluid chromatography and reversed-phase liquid chromatography are compared.
Pavel Jandera, Jan Soukup, Petr Janás, Magda Staňková
In the past ten years, LC on polar stationary phases with mobile phases containing organic solvent and less than 5–40% water (HILIC) has become preferred method for separation of polar compounds, namely pharmaceuticals or biopolymers. On polar columns, sample interactions, retention and separation selectivity may strongly change across the full composition range of aqueous-organic mobile phases, HILIC retention mechanism predominating in the organic-rich, and reversed-phase (RP) mechanism in more aqueous media. We determined the accumulation of water from acetonitrile-rich aqueous-organic mobile phases in the water-rich diffuse layer filling the pores of eighteen polar columns, using frontal chromatography combined with Karl Fischer titration. The sorption can be described by Langmuir isotherm and it strongly depends on the type of stationary phase, ranging from less than one monomolecular water layer up to 6 - 10 water layers equivalents. Some water present in polar solvents is accumulated even on non-polar columns. Considering the actual concentration of water in calculations largely improves the accuracy of prediction of gradient elution data in HILIC systems. The dual retention mechanism enables using a single polar column alternatively in the HILIC and RP retention modes of various polar sample compounds such as phenolic acids and flavones. This approach enhances separation selectivity and peak capacity in single- and two-dimensional HPLC.
One of the most successful approaches to the retention and separation of polar compounds is hydrophilic interaction liquid chromatography (HILIC). HILIC is not a straightforward technique and one of its most common drawbacks during method development is the difficulty in identifying the most suitable column to use. The stationary phases used in HILIC are quite diverse, and systematic studies of their chemistries are relatively limited. Kumar et al.  re-developed early characterisation studies, incorporating the effects of changing a range of experimental parameters as well as a characterisation study on the stationary phases. This approach has been previously applied in RPLC [2, 3], but novel in HILIC and represents a good practical guide to adjustments of conditions that effect the selectivity of a separation.
The aim of this work was to develop these areas of critical understanding by testing a wider range of experimental parameters to determine the interactions between them and how these parameters affect the chromatography of a series of test compunds. Five main classes of HILIC columns were investigated in this study:
Bare silica, neutral bonded ligands, charged ligands, zwitterionic phases and mixed mode phases. A preliminary column characterisation investigation was performed. This highlighted fundamental differences in the column chemistries. In addition the following experimental factors were studied: effect of solvent content, effect of buffer concentration and effect of buffer pH. The work was finally summarised in a convenient work flow which describes in detail a generic approach to method development within the HILIC mode of chromatography.
 Kumar A., Heaton J.C. and McCalley D.V., 2013, J. Chromatogr. A, 1276, 33.
 Neue U. D., O’Gara J.E. and Mendez A., 2006, J. Chromatogr. A, 1127, 161.
 Neue U. D. and Mendez A., 2007, J. Sep. Sci., 30, 949.
Tony Edge,1 Peter Myers,2 Haifei Zhang,2 Adham Ahmed,2 Richard Hayes,2 Luisa Pereira1
1 Thermo Fisher Scientific, Tudor Road, WA7 1TA, Runcorn, UK
In recent years there has been a renewed interest in particle technology for both HPLC and UHPLC, due to the introduction of solid core technology. Prior to this there was very little novel development in HPLC particle morphology The patents associated with solid core technology have resulted in manufacturers and researchers having a much greater level of interest in the basic manufacture of silica particles, particularly since the current manufacturing approach is known to be a fairly involved process.
This has led to the development of a completely new type of particle which is manufactured using a one-step synthesis delivering near monodispersed particles and core shell morphology. The morphology of the particle has been designed to deliver the real advantages of the core-shell particles, with a range of parameters being varied to ensure the robustness of the manufacturing.
Careful investigation of some of the morphologies produced revealed that the structure of the particle produced resembled very closely a true fractal structure. Fractal structures have been associated with chromatographic media for some time, however they have been more associated with the concept of being a fractional dimension rather than having a degree of self similarity. These new particles demonstrate, not only a fractional dimension, but also a degree of self similarity. This opens up a possibility of having a surface topography which is essentially homogeneous to solute molecules, thus eliminating one of the issues associated with traditional porous structures in which molecules effectively do not experience the same surface topography resulting in different retention times, the so called ‘c’ term in the van Deemter equation. This is particularly pertinent for large molecules where diffusional processes related to the different pore depths can result in poor peak shape, either through equilibration effects or because molecules effectively get stuck between the walls of the pores.
Examples will be given of the different morphologies that can be manufactured and also how these can be applied to the analysis of large protein molecules.
Boreskov Institute of Catalysis SB RAS, Novosibirsk, Russian Federation, Pr. Lavrentyeva
Comprehensive two dimensional gas chromatography (GCxGC) is a high-effective technique for analysis of complex multicomponent mixtures.
An important advantage of GCxGC is that it allows analyzing of complex samples by separating the components in groups according to chemical classes. Frequently those samples contain high-boiling fractions of compounds. The examples of those samples are crude oil, products of petrochemistry, coal chemistry and etc. It is known that in order to obtain the best separation power in GCxGC one should use as polar phase as possible for one of the dimension. However, the thermal stability of common polar and high polar stationary phases is not sufficient to analyze high boiling complex samples.
Recent advances in the development of new liquid phases made it possible to use some classes of ionic liquids (ILs) as polar stationary phases . There are works devoted to research of columns with imidazolium and phosphonium ILs for GCxGC in the literature . However, the possibility of using ILs for high temperature GCxGC separations (up to 300°C) is not closely investigated. Our work is devoted to the research of imidazolium and pyridinium ILs as stationary phases columns for GCxGC. In our earlier studies we have shown that these classes of ILs allow to work at the temperature up to 300°C  and possess different range of selectivity . Therefore, we consider that their use in GCxGC will allow to obtain the high selective separations at high temperature. In our work the series of columns with pyridinium and imidazolium ILs were prepared and were investigated as columns for GCxGC. These columns were shown to work at the temperature 280°C-300°C with good separation characteristics. The number of separation of multicomponent high boiling samples was obtained. Among them: crude oil and oil products, natural bitumen, high boiling mixtures of biological origin.
Non-covalent binding of biological molecules controls cellular regulation, it is also pivotal to action of modern drugs and molecular diagnostics of diseases. Therefore, understanding molecular mechanisms of biological processes as well as the development of drugs and design of diagnostic systems require affinity methods suitable for three types of applications: (i) kinetic studies of affinity interactions, (ii) selection of affinity ligands from combinatorial libraries, and (ii) the use of affinity ligands as diagnostic probes in molecular diagnostics of diseases. Our answer to this challenge is Kinetic Separation - a conceptual platform for the development of homogeneous kinetic affinity methods suitable for all three applications. One-dimensional (column) separation of any nature, e.g. gel-filtration, electrophoresis, thermophoresis, sedimentation, that can segregate affinity complexes from the unbound interactants can be used as the basis for kinetic separation. A variety of different detection methods can be employed in kinetic separation including fluorescence and mass-spectrometry (MS); the latter can uniquely facilitate label-free kinetic analysis of biomolecular interactions. In this lecture, we will explain the physical principles underlying kinetic separation and discuss two practical implementations of kinetic separation: (i) Kinetic Capillary Electrophoresis with Laser-Induced Fluorescence detection (KCE-LIF) and (ii) Kinetic Size-Exclusion Chromatography with MS detection (KSEC-MS). The examples for discussion will be: (i) the use of KCE-LIF for selection, kinetic characterization, and analytical use of DNA aptamers and (ii) the use of KSEC-MS for studying kinetics of reversible protein-drug binding. Major limitations of kinetic separation will be outlines to identify the boundaries of its applicability.