Characterisation of RPLC columns packed with porous sub-2 microm particles

Eight commercially available sub-2 microm octadecyl silane columns (C18 columns) have been characterised by the Tanaka protocol. The columns can be grouped into two groups that display large differences in selectivity and peak shape due to differences in hydrophobicity, degree of surface coverage and silanol activity. Measurements of particle size distributions were made using automated microscopy and electrical sensing zone measurements. Only a weak correlation could be found between efficiency and particle size. Large differences in column backpressure were observed. These differences are not related to particle size distribution. A more likely explanation is differences in packing density. In order to take full advantage of 100-150 mm columns packed with sub-2 microm particles, it is often necessary to employ not only an elevated pressure but also an elevated temperature. A comparison between columns packed with sub-2, 3 and 5 microm versions of the same packing indicates potential method transferability problems for several of the columns due to selectivity differences. Currently, the best alternative for fast high-resolution LC is the use of sub-2 microm particles in combination with elevated pressure and temperature. However, as shown in this study additional efforts are needed to improve transferability as well as column performance.     

Separation of natural product using columns packed with Fused‐Core particles

Three HPLC columns packed with 3 μm, sub‐2 μm, and 2.7 μm Fused‐Core (superficially porous) particles were compared in separation performance using two natural product mixtures containing 15 structurally related components. The Ascentis Express^TM C18 column packed with Fused‐Core particles showed an 18% increase in column efficiency (theoretical plates), a 76% increase in plate number per meter, a 65% enhancement in separation speed and a 19% increase in back pressure compared to the Atlantis T3^TM C18 column packed with 3 μm particles. Column lot‐to‐lot variability for critical pairs in the natural product mixture was observed with both columns, with the Atlantis T3 column exhibiting a higher degree of variability. The Ascentis Express column was also compared with the Acquity^TM BEH column packed with sub‐2 μm particles. Although the peak efficiencies obtained by the Ascentis Express column were only about 74% of those obtained by the Acquity BEH column, the 50% lower back pressure and comparable separation speed allowed high‐efficiency and high‐speed separation to be performed using conventional HPLC instrumentation.     

Ultrahigh-pressure liquid chromatography using a 1-mm id column packed with 1.5-microm porous particles

The evolution of chromatography has led to the reduction in the size of the packing materials used to fabricate HPLC columns. The increase in the backpressure required has led to this technique being referred to as ultrahigh-pressure liquid chromatography (UHPLC) when the column backpressure exceeds 10000 psi (approximately 700 bar). Until recently, columns packed with sub-2-microm materials have generally fitted into two classes; either short (less than 5 cm) columns designed for use on traditional HPLC systems at pressures less than 5000 psi (350 bar), or capillary columns (inner diameters less than 100 microm). By using packing materials with diameters <2 microm to fabricate UHPLC columns, there is an increase in efficiency and a decrease in the analysis time that are directly proportional to the size of the packing material. In order to realize and exploit the increase in efficiency, however, the columns must maintain lengths typically associated with analytical columns (15-25 cm). We have packed 1 mm diameter, 150 mm in length columns with 1.5 microm packing material, and evaluated their performance in UHPLC. The pressure required to achieve optimum linear velocities in plots of plate height versus linear velocity was in the vicinity of 1104 bar (16000 psi). The 1.5 microm particle-packed column was compared with the more traditional 150 mm long analytical columns packed with 3 microm materials. This column showed an efficiency that was approximately twice that observed with the 3 microm packed column and a concomitant reduction in the analysis time, theoretically predicted.     

Comparison of sub-2-microm particle columns for fast metabolite ID

The use of sub-2-microm particle columns for fast high throughput metabolite ID applications was investigated. Three LC-MS methods based on different sub-2-microm particle size columns using the same analytical 3 min gradient were developed (Methods A, B, and C). Method A was comprised of a 1.8 microm particle column coupled to an MS, methods B and C utilized a 1.7 microm particle column (BEH 50 x 2.1 mm2 id) and 1.8 microm particle column coupled to a Q-TOF MS. The precision and the separation efficiency of the methods was compared with repeated standard injections (N=10) of reference compounds verapamil (VP), propranolol, and fluoxetine. Separation efficiency and MS/MS spectral quality were also evaluated for separation and detection of VP and its two major metabolites norverapamil (NVP) and O-demethylverapamil (ODMVP) in human-liver microsomal incubates. Results show that 1.8 microm particle columns show similar performance for separation of VP and its major metabolites and comparable spectral quality in MS(E) mode of the Q-TOF instrument compared to 1.7 microm particle columns. Additionally, the study also confirmed that sub-2-microm particle size columns can be operated with standard analytical HPLC but that performance is maximized by integrating column in UPLC method with reduced void volumes. All the methods are suitable for the determination of major metabolites for compounds with high metabolic turnover. The high throughput metabolite profile analysis using 384-well plate format of up to 48 compounds in incubates of human-liver microsomes was discussed.     

Practical comparison of LC columns packed with different superficially porous particles for the separation of small molecules and medium size natural products

Commercial C(18) columns packed with superficially porous particles of different sizes and shell thicknesses (Ascentis Express, Kinetex, and Poroshell 120) or sub-2-μm totally porous particles (Acquity BEH) were systematically compared using a small molecule mixture and a complex natural product mixture as text probes. Significant efficiency loss was observed on 2.1-mm id columns even with a low dispersion ultra-high pressure liquid chromatography system. The Kinetex 4.6-mm id column packed with 2.6-μm particles exhibited the best overall efficiency for small molecule separations and the Poroshell 120 column showed better performance for mid-size natural product analytes. The Kinetex 2.1-mm id column packed with 1.7-μm particles did not deliver the expected performance and the possible reasons besides extra column effect have been proved to be frictional heating effect and poor column packing quality. Different column retentivities and selectivities have been observed on the four C(18) columns of different brands for the natural product separation. Column batch-to-batch variability that has been previously observed on the Ascentis Express column was also observed on the Kinetex and Poroshell 120 column.     

Evaluation and comparison of very high pressure liquid chromatography systems for the separation and validation of pharmaceutical compounds

Chromatography using sub-2 microm particles is becoming increasingly popular due to the potential for increased speed, resolution, sensitivity, and peak capacity. To meet the demand, various vendors have re-engineered traditional LC systems to operate at pressures of up to 15000 psi to accommodate the elevated backpressures associated with using sub-2 microm particles. This report investigates and compares the performance of three very high pressure LC (VHPLC) systems: Waters Acquity, Agilent 1200 SL, and Thermo Accela. Specifications for the pump, autosampler, column compartment, detector, and software for each instrument are presented. To assess the chromatographic performance of the three instruments, method development and validation were performed for three pharmaceutical compounds and the results are compared and discussed. The material presented herein serves to highlight the different features of the VHPLC instruments, and assess their suitability for the analysis of pharmaceutical compounds.     

Characteristics of superficially-porous silica particles for fast HPLC: some performance comparisons with sub-2-microm particles

Columns of 2.7-microm fused-core (superficially porous) Type B silica particles allow very fast separations of small molecules at pressures available in most high-performance liquid chromatography instruments. These highly-purified particles with 1.7-microm solid silica cores and 0.5-microm-thick shells of 9 nm pores exhibit efficiencies that rival those of totally porous sub-2-microm particles but at one-half to one-third of the column back pressure. This presentation describes other operating features of fused-core particle columns, including sample loading characteristics and packed bed stability. The superior mass transfer (kinetic) properties of the fused-core particles result in much-improved separation efficiency at higher mobile phase velocities, especially for > 600 molecular weight solutes.     

The application of small porous particles, high temperatures, and high pressures to generate very high resolution LC and LC/MS separations

The effect of combining sub-2 microm porous particles with elevated operating temperatures on chromatographic performance has been investigated in terms of chromatographic efficiency, productivity, peak elution order, and observed operating pressure. The use of elevated temperature in LC does not increase the obtainable performance but allows the same performance to be obtained in less time. Increasing the column temperature did allow the use of longer columns, generating column efficiencies in excess of 100,000 plates and gradient peak capacities approaching 1000. Raising the temperature increased the optimal mobile phase linear velocity, negating somewhat the pressure benefits observed by reducing the solvent viscosity. When operating at higher temperature the analyte retention is not only reduced, but the order of elution will also often change. High temperature separations allowed exotic organic modifiers such as isopropanol to be exploited for alternative selectivity and faster analysis. Finally, care must be taken when using high temperature separations to ensure that the narrow peak widths produced do not compromise the quality of data obtained from detectors such as high resolution mass spectrometers.     

High throughput liquid chromatography with sub-2 microm particles at high pressure and high temperature

In this study, ultra performance liquid chromatography (UPLC) using pressures up to 1,000 bar and columns packed with sub-2 microm particles has been combined with high temperature mobile phase conditions (up to 90 degrees C). By using high temperature ultra performance liquid chromatography (HT-UPLC), it is possible to drastically decrease the analysis time without loss in efficiency. The stability and chromatographic behavior of sub-2 microm particles were evaluated at high temperature and high pressure. The chromatographic support remained stable after 500 injections (equivalent to 7,500 column volumes) and plate height curves demonstrated the capability of HT-UPLC to obtain fast separations. For example, a separation of nine doping agents was performed in less than 1 min with sub-2 microm particles at 90 degrees C. Furthermore, a shorter column (30 mm length) was used and allowed a separation of eight pharmaceutical compounds in only 40s.     

Recent developments in liquid chromatography--impact on qualitative and quantitative performance

In order to reduce the analysis time and maintain good efficiency in liquid chromatography (LC), several solutions are currently being investigated. The focus of this study was to compare, both qualitatively and quantitatively, the chromatographic performance of a conventional LC with selected approaches, namely monolithic supports, high temperature LC (up to 90 degrees C), and sub-2 microm particles combined with high pressure (up to 1000 bar). This comparison was achieved from a qualitative point of view with a special attention paid to the analysis of time reduction, efficiency improvement, and pressure constraint. For this purpose, the different approaches were discussed using Knox curves and other kinetic plots. It appeared that columns packed with sub-2 microm particles under high-pressure conditions (UPLC) were well adapted and this option represents an attractive alternative to conventional LC; however, the other alternative approaches should not be neglected. The quantitative evaluation of these techniques was performed on the basis of the validation of results of a pharmaceutical formulation (Rapidoca?ne), following SFSTP 2003 guidelines. Fast-LC approaches demonstrated equivalent performance to conventional LC in terms of trueness, precision, and accuracy profile, with a significant time reduction (up to 8x) according to the selected methodology.     

Effects of column length, particle size, gradient length and flow rate on peak capacity of nano-scale liquid chromatography for peptide separations

The effects of the column length, the particle size, the gradient length and the flow rate of a nanoLC system on peptide peak capacity were investigated and compared. Columns packed with 1.7 microm and 3 microm C(18) materials into pieces of 75 microm capillary tubing of various lengths were tested with different gradient lengths and flow rates. While increasing the length of a column packed with the 1.7 microm material helped improve peptide peak capacity at the whole range of the tested gradient lengths (24-432 min), little improvement in peak capacity was observed with the increase of the length of a column packed with the 3 microm material unless a gradient longer than 50 min was carried out. Up to 30% of peak capacity increase was observed when a column's length is doubled, with little reduction in the throughput. In most cases, more than 50% of the increase in peak capacity was obtained with the reduction in the particle size from 3 microm to 1.7 microm. With the same backpressure generated, a shorter 1.7-microm-particle column outperformed a longer column packed with the 3 microm material. In a flow rate range of 100-700 nl/min, increasing the flow rate improved peak capacity for columns packed with 1.7 microm and 3 microm materials.     

Short communication performance of octadecylsilylated monolithic silica capillary columns of 530 microm inner diameter in HPLC

Monolithic silica capillary columns were successfully prepared in a fused silica capillary of 530 microm inner diameter and evaluated in HPLC after octadecylsilylation (ODS). Their efficiency and permeability were compared with those of columns pakked with 5-microm and 3-microm ODS-silica particles. The monolithic silica columns having different domain sizes (combined size of through-pore and skeleton) showed 2.5-4.0-times higher permeability (K= 5.2-8.4 x 10(-14) m2) than capillary columns packed with 3-mm particles, while giving similar column efficiency. The monolithic silica capillary columns gave a plate height of about 11-13 microm, or 11 200-13 400 theoretical plates/150 mm column length, in 80% methanol at a linear mobile phase velocity of 1.0 mm/s. The monolithic column having a smaller domain size showed higher column efficiency and higher pressure drop, although the monolithic column with a larger domain size showed better overall column performance, or smaller separation impedance (E value). The larger-diameter (530 microm id) monolithic silica capillary column afforded a good peak shape in gradient elution of proteins at a flow rate of up to 100 microL/min and an injection volume of up to 10 microL.     

Evaluation of frontal chromatograms

A novel pressure-balanced injection valve was evaluated for use with ultrahigh pressure liquid chromatography (UHPLC) at pressures up to 120 MPa (1,200 bar). Fused-silica capillaries (30-33 cm x 100 microm I.D.) packed with nonporous 1.5 microm isohexylsilane-modified (C6) silica particles were employed to study maximum pressure, injection reproducibility, injection time, and sample amount consumed for an injection. The new valve was more reproducible, convenient, and required much less sample than previously used injection systems. The effect of column diameter on efficiency and sensitivity was studied. The 100 microm I.D. columns demonstrated approximately 40% lower efficiency but 10-fold higher sensitivity than the 29 microm I.D. columns. Columns packed with nonporous C6 particles produced higher efficiencies than columns packed with a 1.5 microm porous octadecylsilane-modified (C18) material.     

Modifying conventional high-performance liquid chromatography systems to achieve fast separations with Fused-Core columns: a case study

The theoretical increase in performance from the use of high efficiency columns with conventional HPLC equipment is generally not observed due to the design limitations of such equipment, particularly with respect to extra-column dispersion (ECD). This study examines the impact of ECD from a Waters Alliance 2695 system on the performance of 2.7 μm HALO C(18) Fused-Core superficially porous particle columns of various dimensions. The Alliance system was re-configured in different ways to reduce extra-column volume (ECV) and the ECD determined in each case as a function of flow rate up to a maximum of 2 mL/min. The results obtained showed a progressive decrease in ECD as the ECV was reduced, irrespective of the flow rate employed. However, this decrease in ECD was less than theoretically expected for the lower ECV configurations. The inability to reduce the actual extra-column dispersion further was attributed to additional dispersion associated with the design/volume of the auto-injector. This was confirmed by making sample injections with a low dispersion manual injection valve, instead of auto-injection, for the two lowest ECV configurations studied. In each case, the measured and predicted ECD values were in good agreement. The auto-injector module is an integral part of the Alliance 2695 instrument and cannot be easily modified. However, even with autosampler injection, for a 3mm ID × 100 mm Fused-Core column approximately 70% of the maximum plate count (～84% of the resolution or more) could still be obtained in isocratic separations for solutes with k ≥ ～4.5 when using the lowest ECV configuration. This study also highlights some of the problems inherent in trying to measure accurately the true extra-column dispersion of a chromatographic system and compares the results obtained to those theoretically predicted. Using this same lowest volume instrument configuration, two real-world pharmaceutical methods were scaled to separations that are ～3-3.5-fold faster, while still maintaining comparable data quality (resolution and signal-to-noise ratios).     

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.

     

Mass transport in sub-2-microm high-performance liquid chromatography

Ultra-high pressure liquid chromatography enables increased separation speed and efficiency. The quantitative improvement in efficiency is lower than that predicted by theory, and the reasons are not known. In this work, slow mass transport due to analyte desorption from the stationary phase is discussed as a possible contribution to the lower than expected efficiency. Data in the literature for the reversed phase elution of acetophenone, for which particle size was varied with constant particle composition and mobile phase, were used to test this possibility. The mass transport terms for the three particles sizes (1.7, 3.5 and 5.0 microm) fit well to a model that includes desorption from the stationary phase as a contribution, and this analysis yields an apparent desorption time constant of 2.0(+/-0.2)ms for acetophenone in a reversed-phase separation. The results indicate that it is reasonable to consider slow desorption as a possible contribution to the reduced plate height for sub-2-microm particles.     

Comparison of LC Columns Packed with 2.6?��m Core-Shell and Sub-2?��m Porous Particles for Gradient Separation of Antibiotics

The recently introduced Kinetex C18 column packed with core-shell 2.6 ??m particles is declared to provide similar efficiency and short analysis as Acquity BEH C18 column with 1.7 ??m porous particles. Unlike Acquity BEH C18 column, Kinetex C18 column exhibited lower column backpressure making this column compatible to conventional LC systems. The performance of Kinetex C18 column (2.1 × 50 mm) and Acquity BEH C18 column (2.1 × 50 mm) for gradient separation of tetracyclines under acidic conditions (oxytetracycline, tetracycline, chlortetracycline, and doxycycline) and macrolides under alkaline conditions (tylosin, clarithromycin, roxithromycin, and carbomycin) was studied. The columns were compared by evaluation of their experimental peak capacity and its dependence on linear velocity and gradient slope. The maximal experimental peak capacities for analysis of tetracyclines were 51.8 (Acquity BEH C18 column) and 48.4 (Kinetex C18 column). This indicated that Kinetex C18 was a suitable alternative to Acquity BEH C18 column for the analysis of tetracyclines under acidic conditions. On the contrary, the maximal experimental peak capacities for analysis of macrolides on Acquity BEH C18 column was higher (46.7) than that on Kinetex C18 column (36.9). Moreover, application of Kinetex C18 column for the analysis of macrolides under alkaline conditions was limited with respect to its decreasing performance with growing number of injections on the column.     

Monolithic silica columns with various skeleton sizes and through-pore sizes for capillary liquid chromatography

Reduction of through-pore size and skeleton size of a monolithic silica column was attempted to provide high separation efficiency in a short time. Monolithic silica columns were prepared to have various sizes of skeletons (approximately 1-2 microm) and through-pores (approximately 2-8 microm) in a fused-silica capillary (50-200 microm I.D.). The columns were evaluated in HPLC after derivatization to C18 phase. It was possible to prepare monolithic silica structures in capillaries of up to 200 microm I.D. from a mixture of tetramethoxysilane and methyltrimethoxysilane. As expected, a monolithic silica column with smaller domain size showed higher column efficiency and higher pressure drop. High external porosity (> 80%) and large through-pores resulted in high permeability (K = 8 x 10(-14) -1.3 x 10(-12) m2) that was 2-30 times higher than that of a column packed with 5-mirom silica particles. The monolithic silica columns prepared in capillaries produced a plate height of about 8-12 microm with an 80% aqueous acetonitrile mobile phase at a linear velocity of 1 mm/s. Separation impedance, E, was found to be as low as 100 under optimum conditions, a value about an order of magnitude lower than reported for conventional columns packed with 5-microm particles. Although a column with smaller domain size generally resulted in higher separation impedance and the lower total performance, the monolithic silica columns showed performance beyond the limit of conventional particle-packed columns under pressure-driven conditions.     

Preparative capillary zone electrophoresis using a dynamic coated wide-bore capillary

Preparative capillary zone electrophoresis separations of cytochrome c from bovine and horse heart are performed efficiently in a surfactant-coated capillary. The surfactant, dimethylditetradecylammonium bromide (2C(14)DAB), effectively eliminated protein adsorption from the capillary surface, such that symmetrical peaks with efficiencies of 0.7 million plates/m were observed in 50-microm id capillaries when low concentrations of protein were injected. At protein concentrations greater than 1 g/L, electromigration dispersion became the dominant source of band broadening and the peak shape distorted to triangular fronting. Matching of the mobility of the buffer co-ion to that of the cytochrome c resulted in dramatic improvements in the efficiency and peak shape. Using 100 mM bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane phosphate buffer at pH 7.0 with a 100-microm id capillary, the maximum sample loading capacity in a single run was 160 pmol (2.0 microg) of each protein.     

Method to predict and compare the influence of the particle size on the isocratic peak capacity of high-performance liquid chromatography columns

A kinetic plot based method has been used to experimentally predict the optimal particle size yielding the maximal isocratic peak capacity in a given analysis time. Applying the method to columns of three different manufacturers and characterizing them by separating a 4-component paraben mixture at 30 degrees C, it was consistently found that the classical 3 and 3.5 microm particles provide the highest peak capacity for typical isocratic separation run times between 30 and 60 min when operating the columns at a conventional pressure of 400 bar. Even at 1000 bar, the sub-2 microm particles only have a distinct advantage for runs lasting 30 min or less, while for runs lasting 45 min or longer the 3 and 3.5 microm again are to be preferred. This finding points at the advantage for high-resolution separations that could be obtained by producing 3 and 3.5 microm particle columns that can be operated at elevated pressures.