Porous Sub-2 Μm Research Articles

Comparison of small size fully porous particles and superficially porous particles of chiral anion-exchange type stationary phases in ultra-high performance liquid chromatography: effect of particle and pore size on chromatographic efficiency and kinetic performance

Recent advancements in particle design are common in reversed-phase liquid chromatography (RPLC), but in chiral separations their use is still sporadic in commercially available chiral stationary phases (CSPs). Due to reported lower mass transfer resistance, they might be a promising opportunity to increase efficiency and reduce time of analysis since the relatively higher mass transfer resistance term of CSPs caused by slow adsorption-desorption kinetics is the most performance-limiting factor in enantioselective chromatography. This study was dedicated to the evaluation of new support materials for tert-butylcarbamoylquinine (tBuCQN) based CSP to provide highly efficient and fast enantioseparations. As the main focus of this study, the chiral selector tBuCQN was immobilized on sub-2 μm fully porous particles (FPPs) and 2.7 μm superficially porous particles (SPPs) and their column performance in enantioseparation was evaluated in comparison to 5 μm FPPs by van Deemter and Knox analyses as well as kinetic plots using racemic Fmoc-Phe. Both new particle types outperformed the 5 μm FPP benchmark in terms of speed and efficiency, with wider pore materials (160 or 200 Å) being advantageous (over 90 or 120 Å). Basically decisive for the performance gain was the 10-times smaller mass transfer resistance. Furthermore, 2.7 μm 160 Å SPPs outperformed their fully porous sub-2 μm 120 Å counterpart (HminR = 4.64 μm vs. HminR = 8.94 μm) due to various parameters affording reduced plate height h of 1.7. Caused by the inaccessible core, separations were about 2-times faster. Packing of 2.7 μm core-shell particles provided a very homogeneous column bed, and, owing to its higher permeability, the column backpressure was much lower. It enables packing of longer columns providing theoretically separation efficiencies of up to 106 plates per m (as indicated by kinetic plots) and versatile use without the necessity of UHPLC systems. Investigating the effect of particle size reduction (FPPs: 5 μm, 3 μm, 1.7 μm; SPPs: 2.7 μm, 2 μm) and wider pores (FPPs: 120 Å, 200 Å; SPPs: 90 Å, 160 Å), a significantly reduced mass transfer resistance was the driving force for performance gain. Individual contributions of peak dispersion were deconvoluted for 5 μm FPP CSP and confirmed that slow adsorption-desorption kinetics is the most significant contribution to peak broadening in this chromatographic system.

Benefits and Drawbacks of Fully Porous sub-2 µm and Core-Shell Particles. Comparative UHPLC Applications in Food, Environmental, Forensic, Biopharmaceutical and Natural Products Analyses

Benefits and Drawbacks of Fully Porous sub-2 µm and Core-Shell Particles. Comparative UHPLC Applications in Food, Environmental, Forensic, Biopharmaceutical and Natural Products Analyses

High speed hydrophilic interaction liquid chromatographic method for simultaneous determination of selected pharmaceuticals in wastewater using a cyano-bonded silica column

ABSTRACTIn this work, a fast analytical method based on hydrophilic interaction liquid chromatographic-Ultraviolet detection (HILIC-UV) using a short narrow bore cyano-bonded silica column packed with fully porous sub-2 µm particles has been developed for simultaneous determination of eight pharmaceuticals in wastewater. The method involved pre-concentration and clean-up by solid phase extraction using Oasis HLB extraction cartridges. The analytes were separated using a mobile phase consisted of acetonitrile and 5 mM ammonium acetate buffer (95:5 v/v) with a flow rate of 0.6 mL/min. The chromatographic separation was optimized in order to achieve short analysis time and good resolution for all analytes in a single run. Each analyte was detected at its maximum wavelength for higher sensitivity. All analytes could be separated in 5.7 min with resolution ≥2.7. The optimized method was validated based on linearity, precision, detection and quantification limits, selectivity and accuracy. The detection limits of the studied pharmaceuticals ranged from 0.6 to 3 µg/L, while limits of quantification were in the range from 2 to 10 µg/L with UV detection. The developed method is fast, reliable, cost-effective and could be used for the analysis of the studied analytes in other matrices such as food, pharmaceutical preparations and biological fluids.

Recent advances in liquid-phase separations for clinical metabolomics.

Over the last decades, several technological improvements have been achieved in liquid-based separation techniques, notably, with the advent of fully porous sub-2 μm particles and superficially porous sub-3 μm particles, the comeback of supercritical fluid chromatography, and the development of alternative chromatographic modes such as hydrophilic interaction chromatography. Combined with mass spectrometry, these techniques have demonstrated their added value, substantially increasing separation efficiency, selectivity, and speed of analysis. These benefits are essential in modern clinical metabolomics typically involving the study of large-scale sample cohorts and the analysis of thousands of metabolites showing extensive differences in physicochemical properties. This review presents a brief overview of the recent developments in liquid-phase separation sciences in the context of clinical metabolomics, focusing on increased throughput as well as metabolite coverage. Relevant metabolomics applications highlighting the benefits of ultra-high performance liquid chromatography, core-shell technology, high-temperature liquid chromatography, capillary electrophoresis, supercritical fluid chromatography, and hydrophilic interaction chromatography are discussed.

Core-shell column Tanaka characterization and additional tests using active pharmaceutical ingredients.

In the last decade, core-shell particles have gained more and more attention in fast liquid chromatography separations due to their comparable performance with fully porous sub-2μm particles and their significantly lower back pressure. Core-shell particles are made of a solid core surrounded by a shell of classic fully porous material. To embrace the developed core-shell column market and use these columns in pharmaceutical analytical applications, 17 core-shell C18 columns purchased from various vendors with various dimensions (50mm × 2.1 mm to 100 mm × 3 mm) and particle sizes (1.6-2.7 μm) were characterized using Tanaka test protocols. Furthermore, four selected active pharmaceutical ingredients were chosen as test probes to investigate the batch to batch reproducibility for core-shell columns of particle size 2.6-2.7 μm, with dimension of 100 × 3 mm and columns of particle size 1.6 μm, with dimension 100 × 2.1 mm under isocratic elution. Columns of particle size 2.6-2.7 μm were also tested under gradient elution conditions. To confirm the claimed comparable efficiency of 2.6 μm core-shell particles as sub-2 μm fully porous particles, column performances of the selected core-shell columns were compared with BEH C18 , 1.7μm, a fully porous column material as well.

Estimation of the effects of longitudinal temperature gradients caused by frictional heating on the solute retention using fully porous and superficially porous sub-2 μm materials

In this study, the retention changes induced by frictional heating were evaluated for model small compounds (150–190Da) and a small protein, namely insulin (5.7kDa). For this purpose, the effect of longitudinal temperature gradient caused by frictional heating was experimentally dissociated from the combined effect of pressure and frictional heating, by working either in constant and variable inlet pressure modes. Various columns packed with core–shell and fully porous sub-2μm particles were tested. It appears that frictional heating was less pronounced on the column packed with smallest core–shell particles (1.3μm), compared to the ones packed with core–shell and fully porous particles of 1.7–1.8μm. This observation was attributed to the low permeability of this material and the fact that it can only be employed in a restricted flow rate range, thus limiting the generated heat power. In addition, the thermal conductivity of the solid silica core of superficially porous particles (1.4W/m/K) is known to be much larger than that of fully porous silica. Then, the heat dissipation is improved. However, if systems with higher pressure capability would be available and the mechanical stability of 1.3μm core–shell material was extended to e.g. 2000bar, the retention would be more severely impacted. At 2000bar, ∼4.4W heat power and +30°C increase at column outlet temperature is expected. Last but not least, when analyzing large molecules, the impact of pressure overcomes the frictional heating effects. This was demonstrated in this study with insulin (∼5.7kDa).

Evolution and Current Trends in Liquid and Supercritical Fluid Chromatography

The current trend in high performance liquid chromatography (HPLC) tends toward the achievement of higher separation efficiency and shorter analysis time. Indeed, better performance in LC has become increasingly important in recent years mainly driven by the challenges of either analyzing more complex samples or increasing the numbers of samples per time unit. In the recent development of particle technology, the use of fully porous sub-2 µm particles and sub-3 µm shell particles have received considerable attention. Beside packed columns, the new generation of silica-based monolithic columns also offers very high separation power. However, to take full advantage of these innovative phases, the chromatographic system has also to be drastically optimized in terms of upper pressure limit and system volume. This revolution in column technology now spreads and covers several modes of liquid chromatography such as reversedphase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), or even supercritical fluid chromatography (SFC). The HILIC and SFC, which can be considered as alternative modes of chromatography, could also be useful to extend the applicability of chromatography towards the analysis of very hydrophilic and lipophilic compounds, respectively. The present review gives an insight about the theory behind the success of current column technology and presents a summary of latest applications, using various modes of one-dimensional chromatography (RPLC, HILIC, SFC). This paper also shows that theoretically expected column efficiency could sometimes be compromised in practical work especially in the case of narrow bore columns.

Ultra-High Performance Liquid Chromatography/Electrospray Ionization-Tandem Mass Spectrometry Determination of 151 Pesticides in Soybeans and Pulses

This paper presents the application of ultra-high performance LC (UHPLC) and MS for the determination of 151 pesticides in soybeans and pulses. A core-shell particle (2.6 micro m particle size) column and a fully porous sub-2 microm (1.7 microm particle size) column showed comparable performance in chromatographic resolution and separation, increasing selectivity, and reducing analysis time. UHPLC was coupled with either a triple quadrupole mass analyzer (MS/MS) or a quadrupole Orbitrap (namely Orbital trap) mass spectrometer (Q-Orbitrap MS), which possesses fast data acquisition capability. Both configurations yielded analytical run times of < or =14 min. Soybean and pulse samples were analyzed and quantitated for pesticide residues using the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) procedure, UHPLC/electrospray ionization (ESI)-MS/MS, and matrix-matched standard calibration curves (in an analytical range of 5-500 microg/kg) with isotopically-labeled standards or a chemical analog as internal standards. The method performance parameters that included overall recovery, intermediate precision, and measurement uncertainty were evaluated according to a nested design experiment. Approximately 89% of the pesticides studied had recoveries between 81 and 110%; 95%, had intermediate precision < or =20%; and 93% showed measurement uncertainty < or =40%. From a pilot study of 100 samples, eight tested positive by UHPLCIESI-MS/MS for carbendazim, methomyl, or imidacloprid. These pesticides were further confirmed using UHPLC/ESI-Q-Orbitrap MS based on accurate mass measurement with mass error < or =5 ppm.

Comparison of a new high-resolution monolithic column with core-shell and fully porous columns for the analysis of retinol and α-tocopherol in human serum and breast milk by ultra-high-performance liquid chromatography†

The reduction of analysis time, cost, and improvement of separation efficiency are the main requirements in the development of high-throughput assay methods in bioanalysis. It can be achieved either by ultra-high-performance liquid chromatography (UHPLC) using stationary phases with small particles (<2 μm) at high back pressures or by using opposite direction--monolithic stationary phases with low back pressures. The application of new types of monolithic stationary phases for UHPLC is a novel idea combining these two different paths. The aim of this work was to test the recently introduced second-generation of monolithic column Chromolith® HighResolution for UHPLC analysis of liposoluble vitamins in comparison with core-shell and fully porous sub-2 μm columns with different particle sizes, column lengths, and shapes. The separation efficiency, peak shape, resolution, time of analysis, consumption of mobile phase, and lifetime of columns were calculated and compared. The main purpose of the study was to find a new, not only economical option of separation of liposoluble vitamins for routine practice.

High resolution reversed phase analysis of recombinant monoclonal antibodies by ultra-high pressure liquid chromatography column coupling

Monoclonal antibodies (mAbs) represent one of the fastest growing areas of new drug development. However, their analytical characterization is complex and generally requires an array of orthogonal analytical techniques. Reversed phase liquid chromatography is a valuable strategy due to its high resolving power and straightforward compatibility to mass spectrometry. The present study demonstrates that high peak capacity can be attained with intact mAb of ∼150kDa, reduced mAb fragments of ∼25–50kDa and also digested mAb generating numerous peptides of ∼0.5–3kDa. Several long columns packed with fully porous wide-pore sub-2μm particles were coupled in series to evaluate the effect of column length on peak capacity. By using three columns of 150mm length, a mobile phase temperature of 80°C and a gradient time of around 20min, peak capacities of 117 and 128 were obtained for a commercial intact mAb and its reduced mAb fragments, respectively. On the other hand, when peptide mapping was performed at 50°C, with a gradient time of 270min and a column length of 450mm, a peak capacity of more than 700 was achieved.

Green ultra‐fast high‐performance liquid chromatographic method using a short narrow‐bore column packed with fully porous sub‐2 μm particles for the simultaneous determination of selected pharmaceuticals as surface water and wastewater pollutants

Fast separations are very desirable in laboratories that analyze large numbers of samples per day or those needing short turn-around times. Traditional HPLC methods using conventional stationary phases and standard column dimensions require significant amounts of organic solvents and generate large volumes of waste. With growing awareness about the environment, the development of green technologies has been receiving increasing attention. In this work, a very fast green analytical method based on LC-UV using a short narrow bore column packed with fully porous sub-2 μm particles has been developed for simultaneous determination of nine pharmaceuticals in wastewater and surface water. The chromatographic separation was optimized in order to achieve short analysis time and good resolution for all analytes in a single run. All analytes could be separated in 1 min with good resolution. Sample preparation was executed by solid phase extraction using Oasis HLB cartridges. The method developed was validated based on parameters such as linearity, precision, accuracy, detection, and quantification limits. The recovery ranged from 70.9 to 92.5% with SDs not higher than 5.4%, except for acetaminophen and sulphanilamide. LODs ranged from 0.6-2.5 μg/L, while the LOQs were in the range 2-8 μg/L.

Fast ultrahigh performance liquid chromatographic method for the simultaneous determination of 25 emerging contaminants in surface water and wastewater samples using superficially porous sub-3 μm particles as an alternative to fully porous sub-2 μm particles

The development of fused-core silica stationary phases was considered a breakthrough in column manufacturing. In this study, a fast LC–UV method was developed and validated for the simultaneous determination of 25 emerging contaminants in surface water and wastewater. The selected analytes belonged to various classes such as veterinary antibiotics, central nervous system stimulants, non-steroidal anti-inflammatory drugs, steroids and preservatives. The chromatographic separation was optimized in order to achieve suitable retention times and good resolution for all analytes in a single run using solvent gradient and flow rate gradient. All analytes eluted within 10min on a Kinetex® C18 column packed with fused core particles. Sample preparation was executed with solid-phase extraction on Oasis HLB cartridges. The method was validated by assessing linearity, selectivity, accuracy, precision, limits of detection and limits of quantification. Good recoveries were obtained for all analytes ranging from 67.5% to 97.0% with standard deviations not higher than 5.7%, except for acetaminophen, sulphanilamide and acetyl salicylic acid, for which lower recoveries were obtained. The detection limits ranged from 1.5 to 15μgL−1, while limits of quantification were in the range from 5 to 50μgL−1. The short analysis time achieved by this method allowed analysis of a large number of samples in a short time, minimizing organic solvent consumption and lowering LC solvents cost. This fast method offers benefits both environmentally and economically.

New trends in reversed-phase liquid chromatographic separations of therapeutic peptides and proteins: Theory and applications

In the pharmaceutical field, there is considerable interest in the use of peptides and proteins for therapeutic purposes. There are various ways to characterize such complex samples, but during the last few years, a significant number of technological developments have been brought to the field of RPLC and RPLC-MS. Thus, the present review focuses first on the basics of RPLC for peptides and proteins, including the inherent problems, some possible solutions and some directions for developing a new RPLC method that is dedicated to biomolecules. Then the latest advances in RPLC, such as wide-pore core-shell particles, fully porous sub-2 μm particles, organic monoliths, porous layer open tubular columns and elevated temperature, are described and critically discussed in terms of both kinetic efficiency and selectivity. Numerous applications with real samples are presented that confirm the relevance of these different strategies. Finally, one of the key advantages of RPLC for peptides and proteins over other historical approaches is its inherent compatibility with MS using both MALDI and ESI sources.

Evaluation of a new wide pore core–shell material (Aeris™ WIDEPORE) and comparison with other existing stationary phases for the analysis of intact proteins

The separation of large biomolecules such as proteins or monoclonal antibodies (mAbs) by RPLC can be drastically enhanced thanks to the use of columns packed with wide-pore porous sub-2μm particles or shell particles. In this context, a new wide-pore core–shell material has been recently released under the trademark Aeris WIDEPORE. It is made of a 3.2μm solid inner core surrounded by a 0.2μm porous layer (total particle size of 3.6μm). The aim of this study was to evaluate the performance of this new material, compare it to other recently developed and older conventional wide-pore columns and demonstrate its applicability to real-life separations of proteins and mAbs. At first, the traditional hmin values of the Aeris WIDEPORE column were determined for small model compounds. The hmin values were equal to 1.7–1.8 and 1.4 for the 2.1 and 4.6mm I.D. columns, respectively, which are in agreement with the values reported for other core–shell materials. In the case of a small protein Insulin (5.7kDa), the achievable lowest h value was below 2 and this impressive result confirms that the Aeris WIDEPORE material should be dedicated to protein analysis. This column was then compared with five other commercially available wide-pore and medium-pore stationary phases, in the gradient elution mode, using various flow rates, gradient steepness and model proteins of MW=5.7–66.8kDa. The Aeris WIDEPORE material often provided the best performance, in terms of peak capacity, peak capacity per time and pressure unit (PPT) and also based on the gradient kinetic plot representation. Finally, real separations of filgrastim (18.8kDa) and its oxidized and reduced forms were performed on the different columns and the Aeris WIDEPORE material provided the most impressive performance (peak capacity>100 for tgrad<6min). Last but not least, this new material was also evaluated on digested and reduced mAb and powerful, high-throughput separations were also attained.

Enhancing the Quality of Separation in One-Dimensional Peptide Mapping Using Mathematical Transformation

In this study, some practical examples are presented that show the quality of separations using very efficient columns packed with the latest generation of core shell sub-3 μm and fully porous sub-2 μm particles in one-dimensional peptide separations. This paper shows an approach for the analysis of proteins, such as high-resolution separations, and a data transformation process to improve peak recognition and analysis. Applying power functions on raw chromatographic data can be a neat tool in the field of biosimilar analysis, especially in comparability studies regarding the quality (primary structure) of proteins. Based on the results presented here, it can be stated that the use of power functions is beneficial for the comparison of chromatograms when peak areas are considered but has no effect when using peak heights. In this study, the new Acquity CSH columns (C18 and phenyl-hexyl) and the core–shell type wide pore Ascentis Express Peptide ES C18 material were applied with great success in peptide mapping. Finally, using phenyl-hexyl stationary phase in peptide separation seems to be a good alternative to the generally applied C18 or C4 phases.

Fused core particles as an alternative to fully porous sub-2 μm particles in pharmaceutical analysis using coupled columns at elevated temperature

The chromatographic performance of columns packed with fused-core particles was compared to that of fully porous particles (sub-2 μm) at elevated temperature and extended column lengths. The study involved a comparison of chromatographic parameters such as retention, selectivity, resolution, efficiency and pressure drop. The analytes showed lower retention on fused core particles compared to sub-2 μm particles at low and high temperatures, with substantially lower backpressure. Van Deemter plots were constructed for both kinds of packing materials using single and coupled columns of different lengths at different temperatures. Lower backpressure across the fused core particles allowed two columns to be coupled at 30 °C to generate ∼66 000 theoretical plates, and three columns could be coupled at 60 °C generating ∼129 000 plates. On the other hand, only two sub-2 μm columns could be coupled at 60 °C generating ∼49 000 plates at the optimum flow rate. Fused-core particles showed similar efficiency to sub-2 μm particles at and below the optimum linear velocity, but with lower backpressure. As an application to pharmaceutical analysis, two mixtures of pharmaceuticals of different classes were separated to illustrate the possibility of using fused-core particles as an alternative to sub-2 μm particles in terms of speed and efficiency. The low back-pressure of the fused-core particles allowed fast separations to be performed without significant loss in efficiency or resolution and allowed column coupling to increase efficiency.

Evaluation of columns packed with shell particles with compounds of pharmaceutical interest

The commercial C18 columns packed with sub-3μm shell particles were tested and compared to a reference UHPLC column, in terms of kinetic performance as well as selectivity, retention capability, peak shape and loading capacity. For this purpose, a set of pharmaceutically relevant molecules was selected, including acidic, neutral and basic drugs. Regarding kinetic performance, hopt values for the shell particles were found between 1.7 and 2, while the UHPLC column provided a value of approximately 2.5. However, this impressive performance should be considered with caution, particularly for the construction of kinetic plots since hopt values were sometimes related to the column dimensions, depending on the provider (hopt comprised between 1.8 and 2.6 for longer columns of 150mm packed with shell particles). Despite the non-porous inner core of the shell particles representing between 25 and 36% of the particle, we demonstrated that the decrease in retention was on the maximum equal to 15% for Ascentis column while Acquity and Poroshell were strictly equivalent in terms of retention. Concerning loading capacity, it remains comparable to that of fully porous sub-2μm particles and always more pronounced with 0.1% formic acid vs. phosphate buffer. The loading capacity of the different columns was found to be better correlated to the pore volume or surface coverage than the shell thickness. Experimentally, the most pronounced overloading was observed with the Poroshell. Finally, the selectivity and peak shape were evaluated using a mixture of basic and acidic drugs. It appears that results were very similar between sub-3μm shell particles and fully porous sub-2-μm particles for our mixture of compounds, showing the ability to transfer existing methods to shell particles, with only limited adjustments. This study confirms the potential of columns packed with shell particles and demonstrates the interest of such column technology with pharmaceutical compounds.

Analysis of peptides and proteins using sub-2μm fully porous and sub 3-μm shell particles

The objective of this study was to evaluate the potential of sub-2 μm totally porous particles and sub-3 μm shell particles for peptide and protein analysis. Specific analytical strategies must be developed for these biomolecules as their importance in the pharmaceutical industry increases and as their structural complexity involves some issues when classical LC conditions are employed. Attention was paid on comparing these different columns in various LC conditions (different temperatures, gradient times, and mobile phase flow rates). The comparison of the different supports was assessed considering columns characteristics (quality of packing, silanol activity, pore size, totally porous or shell particles). In this article, peptides were first analyzed with both column technologies. Similar results to those achieved with low molecular weight compounds were obtained (peak capacity >100 for t(grad) around 3 min and columns dimensions of 2.1 mm id × 50 mm), but specific conditions were required (elevated temperature and the use of a volatile ion-pairing reagent, namely TFA). For peptide analysis following tryptic digestion, the goal was to improve peak capacity and resolution because of the large number of generated peptides. For this purpose, longer columns packed with porous sub-2 μm or shell sub-3 μm particles (i.e., 150 mm) and gradient times (i.e., up to 30 min) were tested. On the other hand, proteins in their intact forms have higher molecular weights (MW>5000 Da) and a tertiary structure, thus requiring different conditions in terms of stationary phase hydrophobicity (C(4)vs. C(18)) and pore size (300 vs. 120 Å). In addition, there were issues with adsorption onto the LC system and/or the column itself. This study showed that proteins with MWs lower than 40,000 Da required chromatographic conditions close to those employed for peptide analysis. For larger proteins, a C(4) 300 Å stationary phase gave the best results, confirming theoretical predictions.

Physical characterization and evaluation of HPLC columns packed with superficially porous particles

Two HPLC columns packed with superficially porous packing material (Kinetex™ 1.7 and 2.6 μm C18 particles) were evaluated in terms of their physical properties and performance characteristics. These columns were compared to a column packed with a sub-2 μm totally porous material and to a Halo(TM) column packed with 2.7 μm C18 superficially porous packing. The columns packed with superficially porous particles displayed a comparably narrower size distribution, which is narrower than the distribution of the totally porous sub-2 μm particles. Physical characteristics of the Kinetex™ particles were evaluated in terms of surface area, pore diameter, and specific pore volume. Total, external, internal, and shell porosities among the four different columns were evaluated and compared. The specific permeability for the Kinetex™ columns showed values close to those predicted by the Kozeny-Carman equation. All four columns were evaluated in terms of their chromatographic performance and compared using the Knox equation. The columns packed with the 2.6 and 2.7 μm superficially porous materials showed reduced plate heights below 2, whereas the sub-2 μm particles showed values of 2.2 and above.

Emerging Analytical Separation Techniques with High Throughput Potential for Pharmaceutical Analysis, Part I: Stationary Phase and Instrumental Developments in LC

In recent years, a trend of change has been observed within pharmaceutical industry. As modern drug discovery has reached a remarkable level of complexity and drugs need to be discovered, developed and produced against strict timelines and within cost- and regulatory constraints, industry seeks "lean" solutions to increase productivity. Among them, increasing the sample throughput of the ever-growing number of necessary (routine) analyses has become a popular target to cut precious time. For the last thirty years, High-Performance Liquid Chromatography (HPLC) has been the leading technology when it comes to various analyses in pharmaceutical industry; however, its necessity of serial analyses taking typically 10-45 min has been a sample throughput-limiting barrier. Lately, the fundamentals of HPLC have been exploited to raise new technologies that can speed up analyses to ground breaking limits, without compromising separation efficiency. This paper reviews some promising technologies, i.e. totally porous sub-2microm particles accompanied by pressures up to 1000 bar (Ultra-Performance Liquid Chromatography or UPLC), fused-core particle technology, monolithic supports and High Temperature Liquid Chromatography (HTLC), having the potential to take LC to the next level in pharmaceutical industry. As each analytical method has its own demands, the advances of the above technologies are discussed for different applications in pharmaceutical analysis where high-throughput analysis can be meaningful, i.e. in a drug discovery and development setting, and in quality operations. Both chemical and biological pharmaceuticals are considered. We discuss the perspectives of these technologies and their realizations up to now in high-throughput pharmaceutical analysis.