Liquid chromatography at the University of Pardubice: A tribute to Professor Pavel Jandera.

Pavel Jandera was a world-leading analytical chemist who devoted his entire professional life to research in the field of high-performance liquid chromatography. During his scientific career, he worked at the Department of Analytical Chemistry at the University of Pardubice, Czech Republic. His greatest contribution to the field of liquid chromatography was the introduction of a comprehensive theory of liquid chromatography with programmed elution conditions. He was also involved in the research of gradient elution techniques in preparative chromatography, modeling of retention and selectivity in various phase systems, preparation of organic monolithic microcolumns, and, last but not least, in the development of theory and practical applications of two-dimensional liquid chromatography, mainly in the comprehensive form. In this review article, we have tried to capture the highlights of his scientific career and provide the readers with a detailed overview of Pavel Jandera's contribution to the evolution of separation sciences.

Kinetic performance comparison of superficially porous, fully porous and monolithic reversed-phase columns by gradient kinetic plots for the separation of protein biopharmaceuticals.

To find the best performing column for the analysis of protein-based biopharmaceuticals is a significant challenge as meanwhile numerous modern columns with distinct stationary phase morphologies are available for reversed-phase liquid chromatography. Especially when besides morphology also several other column factors are different, it is hard to decide about the best performing column a priori. To cope with this problem, in the present work 13 different reversed-phase columns dedicated for protein separations were systematically tested by the gradient kinetic plot method. A comprehensive comparison of columns with different morphologies (monolithic, fully porous and superficially porous particle columns), particle sizes and pore diameters as well as column length was performed. Specific consideration was also given to various monolithic columns which recently shifted a bit out of the prime focus in the scientific literature. The test proteins ranged from small proteins starting from 12 kDa, to medium sized proteins (antibody subunits obtained after IdeS-digestion and disulphide reduction) and an intact antibody. The small proteins cytochrome c, lysozyme and ß-lactoglobulin could be analysed with similar performance by the best columns of all three column morphologies while for the antibody fragments specific fully porous and superficially porous particle columns were superior. A 450 Å 3,5 µm superficially porous particle column showed the best performance for the intact antibody while a 1.7 µm fully porous particle column with 300 Å showed equivalent performance to the best superficially porous column with thin shell and 400 Å pore size for proteins between 12 and 25 kDa. While the majority of the columns had C4 bonding chemistry, the silica monolith with C18 bonding and 300 Å mesopore size approximated the best performing particle columns and outperformed a C4 300 Å wide-pore monolith. The current work can support the preferred choice for the most suitable reversed-phase column for protein separations.

Preparation of a zeolite imidazole skeleton-silica hybrid monolithic column for amino acid analysis via capillary electrochromatography.

We developed a novel, convenient and low-cost one-pot strategy for preparing a zeolitic imidazolate framework-8 (ZIF-8)-silica hybrid monolithic column by adding ZIF-8 directly to a polymer solution of the silica matrix. The simulated stationary phase and monolithic column prepared under optimal conditions were characterized using X-ray diffraction, scanning electron microscopy, Fourier-transform infrared spectroscopy, thermogravimetric analysis nitrogen physisorption and zeta potential. The results obtained confirmed the successful introduction of ZIF-8 into the silica monolithic column, and the prepared monolithic column exhibited good permeability and physicochemical stability. A capillary electrochromatography method was developed based on a ZIF-8-silica hybrid monolithic column through which 15 mixed amino acids, 4 neutral compounds, 4 nipagin esters and 2 chlorinated fungicides were separated in 14, 5, 7 and 6 min, respectively, under optimal conditions. The relative standard deviations retention times and column efficiencies in run-to-run, day-to-day and column-to-column varied in the ranges of 1.90%-2.21%, 2.13%-2.51% and 3.08%-6.65%, respectively, which demonstrated that ZIF-8-silica hybrid monolithic column exhibited satisfactory reproducibility and stability. The incorporation of ZIF-8 into a silica monolithic column is a promising method for preparing novel monolithic columns composed of a metal-organic framework.

Chromatography-Free Purification Strategies for Large Biological Macromolecular Complexes Involving Fractionated PEG Precipitation and Density Gradients.

A complex interplay between several biological macromolecules maintains cellular homeostasis. Generally, the demanding chemical reactions which sustain life are not performed by individual macromolecules, but rather by several proteins that together form a macromolecular complex. Understanding the functional interactions amongst subunits of these macromolecular machines is fundamental to elucidate mechanisms by which they maintain homeostasis. As the faithful function of macromolecular complexes is essential for cell survival, their mis-function leads to the development of human diseases. Furthermore, detailed mechanistic interrogation of the function of macromolecular machines can be exploited to develop and optimize biotechnological processes. The purification of intact macromolecular complexes is an essential prerequisite for this; however, chromatographic purification schemes can induce the dissociation of subunits or the disintegration of the whole complex. Here, we discuss the development and application of chromatography-free purification strategies based on fractionated PEG precipitation and orthogonal density gradient centrifugation that overcomes existing limitations of established chromatographic purification protocols. The presented case studies illustrate the capabilities of these procedures for the purification of macromolecular complexes.

Supercritical fluid chromatography using methacrylate-based monolithic column for the separation of polar analytes.

A poly(N,N-dimethyl-N-methacryloyloxyethyl-N-(3-sulfopropyl) ammonium betaine-co-ethylene dimethacrylate) monolith was prepared in a 100 × 2.0 mm id stainless-steel column and was investigated for supercritical fluid chromatography. Optimization of its porosity was performed by changing the conditions of polymerization. Then, the chemical group of this column was confirmed by Fourier transform infrared spectroscopy and elemental analysis. The morphology was characterized by scanning electron microscopy. The prepared column showed good repeatability based on the retention factor of adenine, uracil and cytosine to calculate their run-to-run, day-to-day, column-to-column, and batch-to-batch relative standard deviations. Those values were less than 1.9% (n = 10), 6.5% (n = 3), 5.6% (n = 3), and 1.7% (n = 3), respectively. In addition, the column was found to be stable within 3 and 10 days with relative standard deviations less than 6.5 and 8.5%, respectively. These results indicated that the columns showed good reproducibility and stability. Compared with liquid chromatogaphy, supercritical fluid chromatography provided better kinetic performance and selectivity. Finally, several neutral, acid, and basic polar analytes were utilized to test its application. The results demonstrated that the prepared column exhibited a good separation performance and showed great potential in supercritical fluid chromatography.

[Fabrication of nanomaterials incorporated polymeric monoliths and application in sample pretreatment].

Polymeric monolithic columns are fabricated by in situ polymerization of the corresponding monomer, crosslinkers, porogenic solvents and radical initiators within a mold. Compared with the conventional packed solid phase extraction adsorbents, polymeric monolithic columns with a continuous porous structure process distinctive advantages of rapid mass transfer and excellent permeability, which facilitates the extraction of trace amounts of the target from the matrix even at high flow velocities. Besides, these materials can be easily fabricated in situ within various cartridges, avoiding a further packing step associated with packed particulate adsorbents. Additionally, the abundant monomer availability, flexible porous structure, and wide applicable pH range make monoliths versatile for use in separation science. Thus, polymeric monolithic columns have been increasingly applied as efficient and promising extraction media for sample pretreatment food, pharmaceutical, biological and environmental analyses. However, these materials usually have the difficulty in morphology control and their interconnected porous micro-globular structure, which may result in low porosity, limited specific surface area and poor efficiency. In addition, polymeric monoliths suffer from the swelling in organic solvents, thus decreasing the service life and precision while increasing the cost consumption. Recently, the development of nanomaterial-incorporated polymeric monoliths with an improved ordered structure, enhanced adsorption efficiency and outstanding selectivity has attracted considerable attention. Nanoparticles are considered as particulates within the size range of 1-100 nm in at least one dimension, which endows them with unique optical, electrical and magnetic properties. These materials have a large surface area, excellent thermal and chemical stabilities, remarkable versatility, as well as a wide variety of active functional groups on their surface. With the aim of exploiting these advantages, researchers have shown great interest in applying nanomaterial-incorporated polymeric monoliths to separation science. Accordingly, significant progress has been achieved in this field. Nanomaterials can be entrapped via the direct synthesis of a polymerization solution that contains well dispersed nanomaterials in porogens. In addition, nanoparticles can be incorporated into the monolithic matrix by copolymerization and post-polymerization modification via specific interactions. Therefore, nanomaterial-incorporated polymeric monoliths combined the different shapes, chemical properties, and physical properties of the polymers with those of the nanoparticles. The presence of nanoparticles can improve the structural rigidity as well as the thermal and chemical stabilities of monolithic adsorbents. Besides, nanoparticles are capable of increasing the specific surface area and providing multiple active sites, which leads to enhanced extraction performance and selectivity of polymeric monolithic materials. In recent years, diverse types of nanomaterials, such as carbonaceous nanoparticles, metallic materials and metal oxides, metal-organic frameworks, covalent organic frameworks and inorganic nanoparticles have been extensively explored as hybrid adsorbents in the modes of solid phase extraction, solid phase microextraction, stir bar sorption extraction and on-line solid phase extraction. This review specifically summarizes the fabrication methods for nanomaterial incorporated polymeric monoliths and their application to the field of sample pretreatment. The existing challenges and future possible perspectives in the field are also discussed.

In situ functionalization of HPLC monolithic columns based on divinylbenzene-styrene-4-vinylbenzyl chloride.

The properties of chromatographic columns are largely determined by functional groups located on the sorbent surface. For monolithic columns, surface functional groups can be created during synthesis stage or by chemical bonding with the complete surface of the sorbent. One of sorbent modification approach is to use on-column click reactions with surface reactive groups. In this study, the surface treatment of monolithic sorbent based on divinylbenzene (DVB), styrene (St) and 4-vinylbenzyl chloride (4VBC) copolymer by heterocyclic nitrogen-containing compounds 1-methylimidazole (1MI), 2-methylimidazole (2MI), 2-methylpyridine (2 MP) and 4-methylpyridine (4 MP) is described. The reaction of nitrogen-containing heterocycles with chloromethyl fragments on the surface results in formation of ion pairs and significantly changes the selectivity of monolithic columns. The chromatographic properties of prepared columns are studied. Modified columns can be operated in reversed-phase (RP) chromatography or in hydrophilic interaction liquid chromatography (HILIC) with different composition of the mobile phase. Separation examples of various chemical substances classes are given.

Fast construction of polymer monolithic columns inside fluorinated ethylene propylene (FEP) tubes for separation of proteins by reversed-phase liquid chromatography.

This paper describes the preparation of polymer monolithic columns in the confines of fluorinated ethylene propylene (FEP) tubes. These tubes are cheap, chemically stable, and widely used in flow analysis laboratories. UV-initiated grafting with 5 wt% benzophenone in methanol for 1 h activated the internal surface walls, thus enabling the further covalent binding of ethylene glycol dimethacrylate (EDMA) from a 15 wt% solution in methanol, also via photografting. Both steps used 254 nm radiation under a potency of 120 mJ cm2. ATR-FTIR measurements revealed the presence of carbonyl, alkyl and vinyl groups in the functionalized FEP. The density of vinyl groups was high enough to firmly attach a poly(lauryl methacrylate-co-ethylene glycol dimethacrylate) monolith in 120 × 1.57 mm i.d. tubes, prepared via photopolymerization. The total preparation lasts less than 2-h. The columns were permeable, (1.58 ± 0.06) × 10-13 m2, providing reproducible chromatographic parameters of retention times, retention factor, selectivity, and resolution. The monoliths were stable at flow rates of 500 µL min-1, collapsing only at flow rates >700 µL min-1, a condition that increased the backpressure over 1000 psi (experiments at the room temperature). The separation of proteins by reversed-phase liquid chromatography demonstrated the efficiency of the columns. Determination of egg white proteins (ovalbumin and lysozyme) and myoglobin in spiked urine proved the applicability to the analysis of real samples.

D-2-allylglycine embedded imidazolium-bridged polyhedral oligomeric silsesquioxane hybrid monolithic column for efficient separation of both small molecules and macromolecules.

The development of multifarious stationary phases is still a growing demand so as to solve the tasks of ever evolving actual applications. Herein, with D-2-allylglycine hydrochloride (AG·HCl) as the hydrophilic monomer, diene ionic liquid 1-allyl-3-vinylimidazolium bromide (AVI·Br) and polyhedral oligomeric silsesquioxane methacryl substituted (POSS-MA) as the dual crosslinkers, the highly cross-linked imidazolium-bridged POSS-AVI-AG hybrid monolithic column was fabricated via the "one-pot" free radical copolymerization. The AG·HCl embedded POSS-AVI-AG column displays typical reversed-phase liquid chromatography/hydrophilic interaction liquid chromatography mixed-mode retention mechanisms. Both hydrophobic phenols, alkylbenzenes, aromatic amines and hydrophilic nucleosides/nucleic acid bases, amides and thioureas were successfully separated with high column efficiencies (up to 571,000 plates/m for amides), outperforming our previously reported AVI·Br modified POSS-AVI column. Moreover, the column was also explored for the separation of cytochrome c tryptic digests and egg white protein extraction. All these results demonstrate that the POSS-AVI-AG column has a good potential in separation of both small molecules and complex biological samples with multiple mechanisms.

Temperature effects on retention and efficiency of butyl and lauryl acrylate porous polymer monoliths in capillary electrochromatography.

The development of organic porous polymer monoliths represents an alternative approach to stationary phase design. The use of these materials has helped to rekindle interest in capillary electrochromatography. Although a large number of investigations have explored different monolith recipes, polymerization conditions, and application challenges, few investigations have addressed the fundamentals of this separation mode with this type of material. This study addresses the thermodynamics of the reversed phase retention mechanism on 100% butyl acrylate and 1:3 butyl:lauryl acrylate (volume/volume ratio) porous polymer monoliths used in a capillary electrochromatography mode. Linear van't Hoff plots yield enthalpies of retention of -3.9 to -14.3 kJ/mol on two different, but related columns for five selected hydrophobic analytes across a thirty degree temperature range. Minimum plate heights were only moderately impacted over this temperature range.