Particle-packed Columns Research Articles

Preparation of monolithic silica HPLC columns with truss-structured skeletons and embedded surfactant-templated mesopores

AbstractMonolithic macro/mesoporous silica gels have been prepared via a sol-gel process using triblock copolymer Pluronic P123 (EO20PO70EO20) as a structure-directing agent. In this synthesis, P123 not only induces phase separation to form macroporous structure but also acts as a supramolecular template to form mesopores with precisely controlled shape and size. Obtained was a monolithic silica composed of continuous truss-like columnar skeletons in which cylindrical mesopores are arranged in a 2D-hexagonal symmetry. These monolithic silica gels have extremely high porosity approaching 90% and exhibited high specific surface area and sharp pore size distribution as revealed by N2 sorption measurements. Combinations of the initial composition and the post-gelation treatment on wet gels allowed the control of physical properties of meso- and macropore structures. The monolithic HPLC columns prepared using these silica gels surface-modified by ODS (octadecylsilyl) ligands gave as many as 140,000 theoretical plates/m for the separation of alkylbenzenes in a reversed-phase mode. Very weak dependence of height equivalent to theoretical plate, H, on the mobile phase velocity was also recognized in comparison with conventional particle-packed columns. Graphical Abstract

Polysaccharide and Cyclodextrin-Based Monolithic Chiral Stationary Phases and its Application to Chiral Separation.

The recent development of monolithic chiral stationary phases (CSPs) for liquid chromatography (LC) is mainly focused on reducing backpressure, maximizing flow rates, faster run time, column efficiency, and stability. This review paper emphasizes recent progress in the development of polysaccharide and cyclodextrin-based monolithic CSPs. Further the paper draws attention to competing techniques, like non-porous particle-packed columns, core-shell and monoliths as chromatographic support matrix, available for achieving fast and efficient chromatographic separation. A brief discussion on the three main classes of chiral monolithic stationary phase viz. silica, organic polymer and hybrid-based monolithic stationary phases is also presented. In addition, the paper highlights various studies on the application of monolith chiral CSPs in LC and capillary electrochromrography separation and analysis of chiral compounds.

Open tubular liquid chromatography: Recent advances and future trends.

Nano-liquid chromatography (nanoLC) is gaining significant attention as a primary analytical technique across various scientific domains. Unlike conventional high-performance LC, nanoLC utilizes columns with inner diameters (i.ds.) usually ranging from 10 to 150μm and operates at mobile phase flow rates between 10 and 1000 nl/min, offering improved chromatographic performance and detectability. Currently, most exploration of nanoLC has focused on particle-packed columns. Although open tubular LC (OTLC) can provide superior performance, optimized OTLC columns require very narrow i.ds.(<10μm) and demand challenging instrumentation. At the moment, these challenges have limited the success of OTLC. Nevertheless, remarkable progress has been made in developing and utilizing OTLC systems featuring narrow columns(<2μm). Additionally, significant efforts have been made to explore larger columns (10-75μm i.d), demonstrating practical applicability in many situations. Due to their perceived advantages, interest in OTLC has resurged in the last two decades. This review provides an updated outlook on the latest developments in OTLC, focusing on instrumental challenges, achievements, and advancements in column technology. Moreover, it outlines selected applications that illustrate the potential of OTLC for performing targeted and untargeted studies.

Longevity and other practical benefits of monolithic silica columns in the analysis of samples with complex matrices.

At the turn of the millennium, the monolithic columns invoked new chances in HPLC. Even more than their organic polymer-based siblings, the inorganic silica-based monoliths targeted the territory of classical fully porous particle-packed columns, promising many benefits. Based on the number of published articles, the monoliths attracted academics just in the first few years after their introduction to the market. Lately, as superficially porous particles and sub-2-micron fully porous particles dominated the market, they stayed in the focus of routine laboratories and those who really appreciated the high porosity of the monolithic bed. The monoliths' practical benefits cannot be easily traced in the literature when they gradually lose academics' interest. Nevertheless, after more than 20 years of our experience, we still favor silica monoliths for their low back pressure and longevity when analyzing samples of clinical, pharmaceutical, and environmental origin. At the same time, the high permeability of monoliths enabled the birth of sequential injection chromatography, the medium-pressure separation technique based on the flexible flow manifold. This minireview aims to check, discuss, and summarize the practical aspects of monolithic silica columns in HPLC and medium-pressure sequential injection chromatography (SIC) that may not be visible at first sight but are evident retrospectively.

Types of High-Performance Liquid Chromatography (HPLC) Columns: A Review

Some chemical analysts, especially those working in the food sector, often find it difficult to choose the HPLC columns for their research. Every so often they tend to modify their method to fit whatever HPLC column available in their laboratory instead of looking for the column type best suited to their experiments. Other than that, HPLC column types are often very limited discussed in the class. This is particularly disadvantageous for those who have sufficient access to select the HPLC column they need for the best result. The lack of insight into the types of HPLC columns available in the market also influenced their decision to select the right column in their analysis to a large extent. This article briefly reviews the differences between the commonly used Particle-Packed Columns with the newer yet less frequently used Monolithic Columns. The types of HPLC columns based on polarity, molecular size, and the electrical charge will be described further, along with the working principle of the columns in brief and various examples of options available on the market. Lastly, the uses of HPLC columns in the food industries are also described. This review is expected to contribute to the field of chemical analysis, such as food analysis.

Eco-Friendly Separation of Antihyperlipidemic Combination Using UHPLC Particle-Packed and Monolithic Columns by Applying Green Analytical Chemistry Principles

Efficient separation of pharmaceuticals and metabolites with the adequate resolution is a key factor in choosing the most suitable chromatographic method. For quality control, the analysis time is a key factor, especially in pharmacokinetic studies. High back pressure is considered as one of the most important factors in chromatography’s flow control, especially in UHPLC. The separation of the anti-hyperlipidemic mixtures was carried out using two columns: a column silica-based particle packed UHPLC and a monolithic column. The systematic suitability of the two columns was compared for the separation of Fenofibrate, its active metabolite, Fenofibric acid and Pravastatin using Atorvastatin as an internal standard. Separation on both columns was obtained using ethanol: buffer potassium dihydrogen orthophosphate pH = 3 (adjusted with orthophosphoric acid) (75:25 v/v) as mobile phase and flow rate 0.8 mL/min. The analytes’ peak detection was achieved by using a PDA detector at 287 nm, 214 nm, 236 nm, and 250 nm for Fenofibrate, Fenofibric acid, Pravastatin, and Atorvastatin, respectively. Reduction of back-pressure was achieved with the monolithic column, where the analytes could be completely separated in less than 1.5 min at a flow rate of 5 mL/min. The principles of Green Analytical Chemistry (GAC) were followed throughout the developed method using environmentally safe solvents.

Ultra-High-Pressure Ion Chromatography with Suppressed Conductivity Detection at 70 MPa Using Columns Packed with 2.5 μm Anion-Exchange Particles.

The use of ultrahigh pressures in combination with columns packed with 2.5 μm microporous and supermacroporous (perfusive) stationary phase particles coated with nanobeads has been successfully explored in ion chromatography with online eluent generation and suppressed conductivity detection. Isocratic separations of inorganic anions and organic acids yielding reduced plate heights as low as 2.1 were achieved, corresponding to efficiencies up to 190000 plates/m, using an optimized system configuration with respect to injection parameters, considering volume and mass loadability, and extra-column dispersion. Viscous-heating effects have been assessed for PEEK-lined stainless steel columns operated at 70 MPa, and effects of thermal gradients on separation efficiency and retention are demonstrated. Whereas the PEEK-lined column hardware acts to some extent as an insulator, a 10% increase in plate number could be obtained when applying a still-air column oven configuration. In the forced-air mode, an increase in retention was observed for polyvalent ions. Finally, the kinetic performance limits of ultrahigh-pressure ion chromatography applying 2.5 μm particle-packed columns operated at 70 MPa were compared to conventional ion-chromatography technology using columns packed with 4 μm particles operated at a maximum pressure of 35 MPa. Downscaling the particle size and increasing the operating pressure led to a maximum time gain with a factor of 3.4, without compromising separation efficiency (N = 10000).

Challenges and Advances in the Fabrication of Monolithic Bioseparation Materials and their Applications in Proteomics Research.

High-performance liquid chromatography integrated with tandem mass spectrometry (HPLC-MS/MS) has become a powerful technique for proteomics research. Its performance heavily depends on the separation efficiency of HPLC, which in turn depends on the chromatographic material. As the "heart" of the HPLC system, the chromatographic material is required to achieve excellent column efficiency and fast analysis. Monolithic materials, fabricated as continuous supports with interconnected skeletal structure and flow-through pores, are regarded as an alternative to particle-packed columns. Such materials are featured with easy preparation, fast mass transfer, high porosity, low back pressure, and miniaturization, and are next-generation separation materials for high-throughput proteins and peptides analysis. Herein, the recent progress regarding the fabrication of various monolithic materials is reviewed. Special emphasis is placed on studies of the fabrication of monolithic capillary columns and their applications in separation of biomolecules by capillary liquid chromatography (cLC). The applications of monolithic materials in the digestion, enrichment, and separation of phosphopeptides and glycopeptides from biological samples are also considered. Finally, advances in comprehensive 2D HPLC separations using monolithic columns are also shown.

Advantages of proteomics using meter‐long monolithic columns with small inner diameter

BackgroundTargeted proteomics by selected reaction monitoring (SRM) with high signal‐noise ratio enables to quantify hundreds of proteins with high sensitivity. However, since the lowest number of proteins is less than 10 copies in a single cell, further improvement from the aspects of sensitivity is necessary to detect these ones.MethodsUnlike conventional particle‐packed columns used in liquid chromatography (LC), monolithic columns are characterized by an orderly 3D‐network structure [1]. With the monolithic columns, high‐performance separation with a thin skeleton and back pressure can be achieved. Further improvement in detection‐sensitivity is achieved by reducing column inner diameter (I.D.), because small elution volume concentrates analytes and efficient desolvation increases ionization efficiency. Hence, we developed meter‐long monolithic columns with small I.D. to SRM‐based proteomics and aimed to improve detection‐sensitivity of mass spectrometry (MS) to increase peak intensity in peptide levels. BSA tryptic digests were directly injected into monolithic columns with different length (500 mm or 1000 mm) and I.D. (100 μm or 75 μm), and peak capacity and peak area were evaluated.Results &amp; DiscussionSeparation efficiency increased 1.9 times by lengthening a monolithic column from 500 mm to 1000 mm. Detection‐sensitivity increased 2.3 times in peptide level by reducing column I.D. from 100 μm to 75 μm. Furthermore, even when column I.D. was reduced, there were no differences in the coefficient of variation values of the peak area. These results showed reduction of I.D. of meter‐long monolithic columns increased detection‐sensitivity in SRM‐based proteomics [2].Support or Funding InformationThis work was supported by JST CREST (Grant Number JPMJCR16G2), Japan.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Comprehensive two-dimensional monolithic liquid chromatography of polar compounds.

Two-dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On-line coupling of a reversed-phase column with an aqueous normal-phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on-line two-dimensional liquid chromatography needs a capillary or micro-bore column providing low-volume effluent fractions transferred to a short efficient second-dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro-columns in fused silica capillaries with structurally different dimethacrylate cross-linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed-phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on-line an organic polymer monolithic capillary column in the first dimension with a short silica-based monolithic column in the second dimension provides two-dimensional liquid chromatography systems with high peak capacities. The silica monolithic C18 columns provide higher separation efficiency than the particle-packed columns at the flow rates as high as 5mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed-phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.

Monolithic alkylsilane column: A promising separation medium for oligonucleotides by ion-pair reversed-phase liquid chromatography

In this paper, a monolithic octadecylsilane column and particle-packed octadecylsilane columns were used to investigate the retention behaviors of oligonucleotides by ion-pair reversed-phase liquid chromatography (IP-RPLC). Results showed that, with same base composition, hairpin oligonucleotides always had weaker retention than corresponding random coil oligonucleotides on the monolithic column, but not on the particle-packed columns. In addition, the linear correlation between the retention factor k of oligonucleotides and the reciprocal of temperature (1/T), especially for hairpins, was relatively weaker on the particle-packed columns, as compared to the correlation on the monolithic column. The correlation between k and 1/T became weaker with decreasing particle size of the particle-packed columns. Moreover, results revealed that the overall retention order on the particle-packed column with small particles (3 μm) differed greatly from that on the monolithic column. In contrast, the retention order on the 10 μm particle-packed column was very close to that on the monolithic column. From the above, we inferred that oligonucleotides could keep their primary conformations unchanged when passing through the monolithic column, attributed to the special pore structures of the monolith. However, the conformations of oligonucleotides were suppressed or even destroyed when oligonucleotides passed through the particle-packed columns. This because the narrow and tortuous channels created by the stacked stationary phase particles could lead to more complex and unequable retention behaviors. Therefore, the monolithic column exhibited better retention regularity for oligonucleotides of secondary structure especially for hairpins than the particle-packed columns. It is noteworthy that the monolith-based IP-RPLC opens an intriguing prospect in accurately elucidating the retention behaviors of oligonucleotides.

Visualizing and studying frictional heating effects in reversed-phase liquid chromatography using infrared thermal imaging

Column temperature control is a fundamental component of liquid chromatography experiments. However, it is typically monitored indirectly by tracking the temperature of an adjacent heating element that exchanges heat with the column in a controlled environment. The practice of not directly measuring the column temperature means that uncontrolled contributions of heat, such as frictional heating inside the column, can be overlooked. The present work describes the use of a high-resolution infrared thermal imaging camera to directly measure the column heat map during mobile phase flow. The approach was used to measure the longitudinal temperature gradient formed with three common mobile phases: water, methanol, and acetonitrile, in two 50 mm reversed-phase columns, a 1.7 μm particle-packed column and a polystyrene divinylbenzene monolith. In a close approximation to an adiabatic environment, the temperature gradients (ΔT) observed with the 1.7 μm particle column at a linear velocity of 5.8 mm/s were up to +16.6 and + 12.8 °C above an ambient temperature of 23 °C for water and acetonitrile, respectively. In the case of water, the measured temperature gradient values (ΔT) were within 1% difference of theoretically-calculated values and on average within 10% for acetonitrile. By contrast, the ΔT observed in the monolith was negligible. The elevated temperatures that are generated through friction in sub-2 μm particle columns may be particularly important to consider for the design of experiments that measure structural features of temperature-sensitive analytes, such as biomolecules. While frictional heating is one important application of the thermal imaging approach described, the technique can be used to provide a data-rich profile of heat exchange in numerous experimental configurations, chromatographic or otherwise.

Multilayered particle-packed column: Evaluation and comparison with monolithic and core-shell particle columns for the determination of red azo dyes in Sequential Injection Chromatography.

A recently presented new type of "multilayered" organic-inorganic hybrid silica particle packed column YMC-Triart C18 (50mm × 4.6mm, 5μm) was used for the development of a sequential injection chromatography method for determination of five azo dyes (Sudan I, Sudan II, Sudan III, Sudan orange G, and para red) in selected food seasonings. The use of a novel sorbent brings attractive features, reduced backpressure, and broader chemical stability together with high separation performance, which are discussed and compared with that of three types of columns typically used in medium-pressure flow chromatography techniques (classic monolithic, narrow monolithic, and core-shell particle columns). The separation was performed in gradient elution mode created by the zone mixing of two mobile phases (acetonitrile/water 90:10, 1.5mL + acetonitrile/water 100:0, 2.3mL) at a flow rate of 0.60mL/min and time of analysis <9.5min. The spectrophotometric detection wavelengths were set to 400, 480, and 500nm. The high performance of the developed method with multilayered particle column was well documented and the results indicate a broad capability of sequential injection chromatography.

Capillary electrochromatography of proteins and peptides (2006-2015).

This review summarizes the development and the applications of capillary electrochromatographic techniques pertinent to the analysis of proteins and peptides, which took place over the last 10 years (2006-mid-2016). This "hybrid" technique is useful for the analysis of a broad spectrum of proteins and peptides and is an approach that is complementary to the liquid chromatographic and the capillary electrophoretic analysis. All modes of capillary electrochromatography are described here, which includes particle-packed columns, monolithic stationary phases, and open-tubular capillary electrochromatography.

Recent Advances and Uses of Monolithic Columns for the Analysis of Residues and Contaminants in Food

Monolithic columns are gaining interest as excellent substitutes to conventional particle-packed columns. These columns show higher permeability and lower flow resistance than conventional liquid chromatography columns, providing high-throughput performance, resolution and separation in short run times. Monoliths possess also great potential for the clean-up and preparation of complex mixtures. In situ polymerization inside appropriate supports allows the development of several microextraction formats, such as in-tube solid-phase and pipette tip-based extractions. These techniques using porous monoliths offer several advantages, including miniaturization and on-line coupling with analytical instruments. Additionally, monoliths are ideal support media for imprinting template-specific sites, resulting in the so-called molecularly-imprinted monoliths, with ultra-high selectivity. In this review, time-saving LC columns and preparative applications applied to the analysis of residues and contaminants in food in 2010–2014 are described, focusing on recent improvements in design and with emphasis in automated on-line systems and innovative materials and formats.

Phospholipid-Modified ODS Monolithic Column for Affinity Prediction of Hydrophobic Basic Drugs to Phospholipids

A phospholipid-modified octadecyl silica (ODS) monolithic column was prepared and its interaction with basic hydrophobic drugs was studied. These drugs are of interest in pharmaceutical research because of their strong interaction with biomembranes. The amount of dimyristoylphosphatidylcholine trapped on the ODS surface was reproducible, and the results of the trinitrobenzenesulfonic acid assay suggested the formation of a monolayer on the surface. Both hydrophobic and electrostatic interactions acted between the model drugs and the phosphatidylcholine. The column was stable for 10 days at least. The column was applied to the affinity screening of basic drugs to phospholipid. Good correlation was obtained between log k and log P for the basic drugs lidocaine, quinidine, propranolol, imipramine, and chlorpromazine. The monolithic silica column allowed highly hydrophobic basic drugs such as imipramine and chlorpromazine to be assayed; these are difficult to analyze by using a conventional particle-packed column. These drugs were clearly separated from acidic drugs naproxen and warfarin on the log k versus log P plots. The thermodynamic studies revealed that the retention of the drug was an enthalpy-driven process, and that the decrease in enthalpy for the phospholipid-modified ODS monolithic column was larger than those for immobilized artificial membrane columns. Our results suggest that the phospholipid-modified ODS monolithic column is applicable to affinity screening of drugs to phospholipids.

Determination of free cortisol and free cortisone in human urine by on-line turbulent flow chromatography coupled to fused-core chromatography–tandem mass spectrometry (TFC–HPLC–MS/MS)

Urinary free cortisol and urinary free cortisone are decisive markers for the diagnosis of syndromes related to the dysfunction of the adrenal gland or to evaluate certain enzymatic disorders. Here, we present a new method, designed for routine laboratory use, which enables quick determination of these analytes with minor sample workup. Turbulent flow chromatography shortens sample preparation, and connection to a fused-core particle-packed column (rugged amide-embedded C18 phase) permits a rapid and effective separation of the analytes, as well as additional separation from other related and isobaric compounds present in urine. Urinary isobaric compounds were successfully identified. The method requires only 100μl of urine supernatant per sample. The total time between injections is 9.5min. The solvents used for both turbulent and analytical chromatography are water and methanol, and the relatively low flows needed during the method resulted in an extended life of the columns. Linearity showed a R (2) > 0.994. Limit of detection and limit of quantification are 0.5 and 1.0ng/ml for cortisone and 1.0 and 2.0ng/ml for cortisol. Recoveries ranged from 99.7 to 109.1% for cortisone and from 98.7 to 102.9% for cortisol. Accuracy values (relative errors) for intra- and inter-assay experiments were always below 8%, whereas precision (percent CV) ranged from 3.7 to 10.7%. No matrix effects were detected during the validation process. The reproducibility for each analyte's retention time was excellent, with a coefficient of variation always below 0.2%. The final validation step included the study of urine samples from healthy children and from children previously diagnosed with corticoidal disorders. The high selectivity achieved enables quick data handling.

Organic monoliths for high‐performance reversed‐phase liquid chromatography

RPLC is the most common mode of LC. It is widely used for separations of both small and large molecules. Monolithic columns, which are currently under intensive study by many groups, have the potential of becoming attractive alternatives to particle-packed columns. They are generally easier and faster to fabricate, and they demonstrate a lower pressure drop, less nonspecific adsorption, and richer chemistry (in the case of organic polymer monoliths) for providing broad selectivity. Silica monoliths, as is also true for columns packed with silica particles, are best applied to small-molecule separations. Organic polymer monoliths, on the other hand, have shown advantages for large-molecule separations. Recently, improvements in organic monoliths have led to efficiencies for small molecules that are approaching and even surpassing 100,000 plates/m. While this performance is still far short of what is currently available using modern small particles and silica monoliths in RPLC, steady progress is being made. This review describes recent developments in the synthesis and performance of organic polymer RPLC monoliths, and identifies areas where additional work is needed to significantly improve their performance for both small- and large-molecule separations.

Chiral Separation of Basic Compounds on Sulfated β-Cyclodextrin-Coated Zirconia Monolith by Capillary Electrochromatography

E-mail: jhpark@ynu.ac.krReceived January 28, 2013, Accepted March 24, 2013Sulfated β-cyclodextrin (SCD)-coated zirconia monolith was used as the chiral stationary phase in capillaryelectrochromatography for enantiomeric separation of basic chiral compounds. SCD adsorbed on the zirconiasurface provided a stable chiral stationary phase in reversed-phase eluents. Retention, chiral selectivity andresolution of a set of six basic chiral compounds were measured in eluents of varying pH, composition ofmethanol and buffer. Optimum mobile phase condition for the separation of the compounds was found to bemethanol content of 30%, buffer concentration of 30 mM and pH of 4.0.Key Words : Chiral separation, Capillary electrochromatography, Zirconia monolith, Sulfated β-cyclodextrinIntroductionCapillary electrochromatography (CEC) on monolithiccolumns are becoming attractive alternative to particle-packed columns in HPLC and electrochromatography.

Monolithic columns in liquid phase separations

The separation of chemical entities in a complex sample mixture is a common problem in chemical/biochemical analysis. Since the vast majority of sample mixtures exist in solution or are brought into solution, liquid phase separations have become a preferred choice for resolving sample mixtures. Within this context and in its broader sense, liquid chromatography (LC) continues to be an indispensable technique in most analytical/bioanalytical laboratories. This is the result of numerous theoretical and technological advances, which have had a remarkable impact on chemical analysis—one recent example is the field of proteomics, which relies heavily on LC to separate complex mixtures of peptides from proteolytic digests. Indeed, it is difficult to imagine an analytical laboratory without state-of-the-art LC equipment. Column technology is one of the most investigated research topics in chromatographic techniques, and LC is not an exception. This is not surprising since it is within the column that the chromatographic processes occur, making the column the “heart” of the separation system. The development of new separation media is essential in achieving a high degree of selectivity, high efficiency, and faster separations to deal with the increasing demands imposed by the complexity of samples in chemical analysis. In recent years, we have seen a remarkable evolution in column technology for LC. Although columns packed with silica microparticulates have predominated in LC and ancillary techniques, monolithic columns have emerged as a viable alternative for liquid phase separations. A monolithic column can be defined as a continuous solid matrix that is porous in nature, containing interconnected flow paths, and can take various formats (e.g., rod-like, flat bed, tubular); the retentive phase is at the surface of this material. The porous, rod-like structures have been the most popular. These porous structures are generally classified into polymeric or silica-based monoliths and can be fabricated by in situ polymerization of organic monomeric precursors in porogenic solvents, for polymeric monoliths, or by sol–gel chemistry in the case of silica monoliths. The main attraction of monolithic columns compared with particle-packed columns is their high axial permeability. This provides low back pressures with excellent flow characteristics, fast mass transfer, and fast separations, without sacrificing separation efficiency. After about two decades of developmental progress since the initial seminal work of Hjerten (1989), Svec (early 1990s), and Tanaka (mid 1990s), monolithic columns have found applicability in different separation modes: reversed-phase chromatography, ion-exchange chromatography, hydrophilic interaction chromatography, size-exclusion chromatography, molecular imprints, and affinity chromatography, to mention a few, and a variety of such modes are commercially available. Monoliths have been used in various formats beyond LC, including solid-phase extraction, sample preconcentration, and 96-well disc arrays for the separation of many compounds in, for example, the pharmaceutical, environmental, microfluidics, and molecular bioprocessing fields. Today, developmental work and applications of monolithic columns in liquid phase separations continue. A quick search in the Web of Knowledge (Thomson Published in the topical collection Monolithic Columns in Liquid Phase Separations with guest editor Luis A. Colon.