Ivo Nischang

On the separation of small molecules by means of nano-liquid chromatography with methacrylate-based macroporous polymer monoliths.

Macroporous monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) stationary phases were synthesized in the confines of 100 microm I.D. fused-silica capillaries via a free radical copolymerization of mono and divinyl monomeric precursors in the presence of porogenic diluents. These columns were used in order to determine their suitability for the reversed-phase separation of small molecules in isocratic nano-LC mode. Carefully designed experiments at varying realized phase ratio by a terminated polymerization reaction, as well as content of organic modifier in the mobile phase, address the most significant parameters affecting the isocratic performance of these monoliths in the separation of small molecules. We show that the performance of methacrylate-based porous polymer monoliths is strongly affected by the retention factor of the analytes separated. A study of the porous and hydrodynamic properties reveals that the actual nature of the partition and adsorption of the small analyte molecules between mobile and stationary (solvated) polymer phases are most crucial for their performance. This is due to a significant gel porosity of the polymeric stationary phase. The gel porosity reflects stagnant mass transfer zones restricting their applicability in the separation of small molecules under conditions of strong retention.

Porous polymer monoliths: morphology, porous properties, polymer nanoscale gel structure and their impact on chromatographic performance.

Porous monoliths based on organic precursors undergoing free-radical cross-linking polymerization in porogenic solvents emerged approximately two decades ago as an alternative stationary phase material for diverse applications including liquid chromatography. Though having a profound difference in morphology to their earlier generation polymer bead-based counterparts, they are often based on similar chemistries and as such show certain peculiarities with respect to transport and performance in liquid chromatography applications. Polymer monoliths typically consist of a globule-like, three-dimensionally adhered backbone, which is in a contrast to the silica monoliths having a bi-continuous mesoporous skeleton. Both material types possess large flow-through pores making them desirable for high performance liquid chromatography and other flow-through applications. The current review is devoted to a critical appraisal of the major challenges that researchers face in the retrieval of the never-ending demand of efficiency at often forgotten and desired selectivity and retention in separations using porous polymer monoliths. Therefore, an attempt is made to establish profound links of polymer monoliths to their earlier generation polymer-based particulate beds and differences to silica-based materials. These links are associated with an emerging morphological understanding of the polymer monoliths porous flow-through pore structure, the nanoscale backbone chemistry, and related chromatographic performances in both theoretical and experimental studies. Associated with this understanding, existing attempts in improving flow and transport performance of polymer monoliths are described and discussed. Such developments are addressing morphological concerns with respect to homogeneity and detailed design of pore space, but also tailoring backbone nanostructural chemistry to modulate mass transfer.

Porous polymer monoliths for small molecule separations: advancements and limitations

Porous polymer monoliths are considered to be one of the major breakthroughs in separation science. These materials are well known to be best suited for the separation of large molecules, specifically proteins, an observation most often explained by convective mass transfer and the absence of small pores in the polymer scaffold. However, this conception is not sufficient to explain the performance of small molecules. This review focuses in particular on the preparation of (macro)porous polymer monoliths by simple free-radical processes and the key events in their formation. There is special focus on the fluid transport properties in the heterogeneous macropore space (flow dispersion) and on the transport of small molecules in the swollen, and sometimes permanently porous, globule-scale polymer matrix. For small molecule applications in liquid chromatography, it is consistently found in the literature that the major limit for the application of macroporous polymer monoliths lies not in the optimization of surface area and/or modification of the material and microscopic morphological properties only, but in the improvement of mass transfer properties. In this review we discuss the effect of resistance to mass transfer arising from the nanoscale gel porosity. Gel porosity induces stagnant mass transfer zones in chromatographic processes, which hamper mass transfer efficiency and have a detrimental effect on macroscopic chromatographic dispersion under equilibrium (isocratic) elution conditions. The inherent inhomogeneity of polymer networks derived from free-radical cross-linking polymerization, and hence the absence of a rigid (meso)porous pore space, represents a major challenge for the preparation of efficient polymeric materials for the separation of small molecules.

Advances in the preparation of porous polymer monoliths in capillaries and microfluidic chips with focus on morphological aspects

Porous polymer monoliths have emerged as unique materials for many applications, including liquid-chromatographic analyses at an unrivaled speed, solid-phase extraction, and enzyme immobilization in capillary and microfluidic chip format. This article reviews the state of the art in the preparation of monoliths in narrow-bore capillaries and microfluidic chips and their miniaturization under conditions of spatial confinement. New developments in their preparation mainly using free radical polymerization techniques with a focus on morphological aspects in view of homogeneous porous materials are described. The suitability of monoliths for analysis of both large and small molecules is also discussed.

On the chromatographic efficiency of analytical scale column format porous polymer monoliths: Interplay of morphology and nanoscale gel porosity

Porous monolithic poly(styrene-co-divinylbenzene) stationary phases in 4.6 mm I.D. analytical-scale column format with varying porosity, globule scale polymer morphology and flow-through pore structure have been investigated with respect to their transport properties toward small retained solutes in isocratic elution, reversed-phase liquid chromatography. The current study was performed under kinetically and thermodynamically relevant conditions comprising retention factors from close to zero up to the order of 50-100 under most extreme conditions, while a linear chromatographic flow velocity up to 4mm/s, in some instances up to 7 mm/s, was realized. Carefully designed experiments aimed at resolving issues associated with the monoliths performance, while a particular focus is given on gel porosity, chromatographic retention and band dispersion. Elucidation of three important metric properties gave orthogonal insight. These are: (i) the columns dry-state morphology and surface area, (ii) the gel porosity with tetrahydrofuran as solvent determined by size exclusion chromatography using a range of small subnanometer-sized molecules and polystyrene standards, as well as (iii) the isocratic reversed-phase performance of small molecules at varying binary acetonitrile/water mobile phase solvent compositions, modulating gel porosity. Consistently throughout the study, the adjustable and general retention-factor-dependence of the performance of these monolithic materials is shown. It can also be correlated to the analytes molecular weight and consequently size. Isocratic performance strongly depends on the amount of gel porosity of the scaffold, which can be changed by varying the percentage of organic modifier in the mobile phase and indicates the adjustable chromatographic nature of porous polymer monoliths. This gel porosity which is absent in the dry-state of the polymer monoliths and is characterized by sub-nanometer to nanometer-sized pore space induces, additionally to permanent porosity, stagnant mass transfer zones. The displayed major reason for mass transfer resistance implied by the use of polymeric monolithic columns determines dispersion behavior of small molecules and its varying importance with respect to morphology and size of the globular features containing stagnant mass transfer zones is addressed. This leads to the conclusion, that a reduction in polymer feature size and increase in number of flow-through pores per unit cross-section of the monolith with an improved homogeneity may be an interesting option of tailoring column performance. It is further concluded that dry-state methods (such as nitrogen adsorption analysis and scanning electron microscopy) or solvated-state methods (such as size-exclusion chromatography in tetrahydrofuran) by itself are insufficient measures to explain the adjustable chromatographic performance of porous polymer monoliths.

Downscaling limits and confinement effects in the miniaturization of porous polymer monoliths in narrow bore capillaries

Monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) columns have been prepared in capillaries ranging in inner diameter from 5 to 75 microm using thermally initiated free-radical polymerization of a mixture of butyl methacrylate, ethylene dimethacrylate, and porogens at different temperatures. Scanning electron microscopy and the measurement of hydrodynamic properties reveal that the downward scalability of the monolithic columns is greatly affected by the confinement effect of the capillary wall resulting from the decreased volume-to-surface ratio as the capillary diameter is decreased. The downscaling process is affected most by the polymerization temperature, the diffusion of the propagating radicals, and the density of coverage of polymerizable groups on the inner walls of the capillary. Optimization of all these factors enables the preparation of monolithic structures in capillaries with inner diameters as low as 5 microm while retaining the desirable properties of monoliths prepared in much larger capillaries. Under these conditions, formation of undesired dense polymer layers attached to the capillary wall was minimized. The chromatographic performance of 10, 25, and 50 microm capillaries evaluated in the reversed phase gradient separation of three proteins showed no change in elution times at identical flow velocities and gradient times, while peak elution width was the smallest with the narrowest capillary.

Effect of capillary cross section geometry and size on the separation of proteins in gradient mode using monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) columns

Porous polymer monoliths have been prepared in capillaries with circular or square cross-sections and lateral dimensions of 50, 75, 100 microm as well as in a rectangular 38 microm x 95 microm capillary. These capillaries have been used to determine the effect of the size and shape of their cross-section on the porous and hydrodynamic properties of poly(butyl methacrylate-co-ethylene dimethacrylate) monoliths. The capillaries were studied by scanning electron microscopy and evaluated for their permeability to flow and their performance in the liquid chromatographic separation of a protein mixture comprising ribonuclease A, cytochrome c, myoglobin, and ovalbumin using a linear gradient of acetonitrile in the mobile phase. No differences resulting from channel geometry were found for the various capillary columns. These results demonstrate that standard capillaries with circular geometry are a good and affordable alternative conduit for modeling the processes carried out in microfluidic chips with a variety of geometries.

Assessing the Nanoscale Structure and Mechanical Properties of Polymer Monoliths used for Chromatography

Concerning polymeric monolithic materials utilized in separation science, the structural and mechanical characteristics from the nanoscopic to the macroscopic scale remain of great interest. Suitable analytical tools are urgently required to understand the polymer monoliths constituent structure, particularly in the case of nanoscale polymer properties that tend to develop gel porosity in contact with a mobile phase ultimately affecting the chromatographic performance. Herein described are our first findings from a characterization of commercially available analytical polymer monoliths based on styrene/divinylbenzene and methacrylate chemistries utilizing confocal Raman spectroscopy imaging and atomic force microscopy (AFM). Confocal Raman spectroscopy can be used to generate a three-dimensional representation of monoliths in both dry state and in contact with solvent. AFM force-indentation measurements on individual cross-sectioned globular features permit detailed assessment of mechanical properties of the stationary phase. This approach allowed so far unprecedented insight and identification of a heterogeneous cross-link density distribution of polymer material within individual globular features on a submicrometer scale.

Multifunctional and biodegradable polyphosphazenes for use as macromolecular anti-cancer drug carriers

Using a living cationic polymerisation procedure we synthesised a series of multi-armed poly(organo)phosphazenes with controlled molecular weights and excellent aqueous solubility. The synthetic flexibility of polyphosphazenes was exploited in order to incorporate an acid-sensitive hydrazide linker to the polymer backbone, as well as tumour-targeting folic acid groups. We were then able to attach hydrophobic anti-cancer drug molecules via the pH labile linker and studied its pH-triggered release kinetics from the polymeric carrier. Although stable for short periods (several days) in an aqueous environment, the polymers were shown to degrade over longer periods (weeks) under simulated physiological conditions. Furthermore, the rate of degradation could be tailored through careful selection of substituents. These biodegradable, multi-functional polyphosphazenes represent promising candidates for use as macromolecular carriers for the tumour-targeted delivery of anti-cancer drugs.

Deviceless decoupled electrochemical detection of catecholamines in capillary electrophoresis using gold microband array electrodes

Samples containing νM concentrations of dopamine, (±)‐isoproterenol, para‐aminophenol and chlorogenic acid have been separated by capillary electrophoresis (CE) and detected using end‐column amperometric detection based on a novel decoupling method. The present decoupling approach involves the use of an electrochemical detector chip containing an array of microband electrodes where the working and reference electrodes are positioned only 10 νm from each other. The short distance between the working and reference electrodes ensures that both electrodes are very similarly affected by the presence of the CE electric field. With this method, no shift in the detection potential was seen when the CE high voltage was applied. This eliminated the need for a reoptimization of the detection potential to compensate for the influence of the separation voltage on the detection. It is also demonstrated that catecholamines can be detected using gold microband electrodes by careful adjustment of the detection potential to avoid the formation of gold oxide. Such careful adjustments of the detection potential are straightforward using the present decoupling method.