Supercritical Fluid Chromatography Instrument Research Articles

Comprehensive Single-Platform Lipidomic/Metabolomic Analysis Using Supercritical Fluid Chromatography-Mass Spectrometry.

Supercritical fluid chromatography (SFC) is a rapidly expanding technique in the analysis of nonpolar to moderately polar substances and, more recently, also in the analysis of compounds with higher polarity. Herein, we demonstrate a proof of concept for the application of a commercial SFC instrument with electrospray ionization-mass spectrometry (MS) detection as a platform for the comprehensive analysis of metabolites with the full range of polarities, from nonpolar lipids up to highly polar metabolites. The developed single-platform SFC-MS lipidomic/metabolomic method is based on two consecutive injections of lipid and polar metabolite extracts from biphase methyl tert-butyl ether extraction using a diol column and two different gradient programs of methanol-water-ammonium formate modifier. Detailed development of the method focused mainly on the pressure limits of the system, the long-term repeatability of results, and the chromatographic performance, including optimization of the flow rate program, modifier composition and gradient, and injection solvent selection. The developed method enabled fast and comprehensive analysis of lipids and polar metabolites from plasma within a 24 min cycle with two injections using a simple analytical platform based on a single instrument, column, and mobile phase. Finally, the results from SFC-MS analysis of polar metabolites were compared with widely established liquid chromatography MS analysis in metabolomics. The comparison showed different separation selectivity of metabolites using both methods and overall lower sensitivity of the SFC-MS due to the higher flow rate and worse chromatographic performance.

Evaluation of control of additive concentration in gradient analysis of supercritical fluid chromatography-coupled to tandem mass spectrometry

Mobile phase additives are used to improve retention behavior in chromatography. In supercritical fluid chromatography (SFC), for which supercritical fluid carbon dioxide (SF-CO2) is used as the main mobile phase, additives can only be added into the modifier. For that reason, when gradient analysis is performed by changing the modifier ratio to SF-CO2, the additive concentration in the mobile phase increases in parallel with the modifier ratio. In a preliminary study performed using the conventional SFC system, ammonium acetate was necessary to improve the peak shape of a polar steroid, dehydroepiandrosterone sulfate (DHEA-S), while the peak intensity of a non-polar steroid, progesterone, decreased by 78% compared to that in the absence of the additive in mobile phase when gradient elution was performed. Since ammonium acetate had both favorable and unfavorable effects on sensitive and simultaneous analysis of these two steroid compounds, a compromise between these effects had to be sought. A three-pump configuration of SFC was developed by adding a pump unit to SFC instrument, which enabled control of the additive concentration independently of the modifier ratio, for the purpose of investigating the additive effect in detail using both steroids as model compounds. The putative cause of the decrease in peak intensity of progesterone was excessively elevated additive concentration in gradient analysis. When the additive concentration in the mobile phase was controlled to ensure that it did not increase during gradient analysis, the peak intensities of progesterone, cortisol, corticosterone, and testosterone were 55%, 40%, 25%, and 17% higher than when the additive concentration was not controlled, respectively. On the other hand, the peak intensity of DHEA-S was almost identical between the conditions, with an increase of 2% with three-pump instrument. The three-pump configuration showed the potential to solve problems relating to the use of modifier additives by keeping their concentration constant in gradient SFC analysis.

Diffusion coefficients of an extensive set of pharmaceutical compounds in supercritical fluid chromatography over a wide range of mobile phase compositions

Diffusion data are essential for adequate analysis of the kinetic separation performance of any chromatographic system. Unfortunately, for Supercritical Fluid Chromatography (SFC), very little data is available of the diffusion coefficients in mobile phases typically used in contemporary methods, i.e. with a non-negligible amount of polar modifier such as methanol. In this work, a relative simple method which only requires minor modifications to a standard commercially available SFC instrument is used to determine the diffusion coefficient of an extensive set of pharmaceutical compounds in the range of 10–50 vol% of modifier (methanol) in CO2. By using a traditional SFC column, the solute is first separated from the sample solvent plug, before entering a long capillary, where the band broadening can be linked to its diffusion coefficient using the Taylor-Aris equation. By using two UV-detectors, before and after the capillary, the effect of the dispersion in the column can be eliminated and the true volumetric flow rate determined. It was found that in the investigated range of conditions, the change in mobile phase viscosity in a first approximation allows to predict the variation in diffusion coefficient. Chemical structure and more particularly functional groups can however have a significant effect on the diffusion coefficient.

Interlaboratory study of a supercritical fluid chromatography method for the determination of pharmaceutical impurities: Evaluation of multi-systems reproducibility

Modern supercritical fluid chromatography (SFC) is now a well-established technique, especially in the field of pharmaceutical analysis. We recently demonstrated the transferability and the reproducibility of a SFC-UV method for pharmaceutical impurities by means of an inter-laboratory study. However, as this study involved only one brand of SFC instrumentation (Waters®), the present study extends the purpose to multi-instrumentation evaluation. Specifically, three instrument types, namely Agilent®, Shimadzu®, and Waters®, were included through 21 laboratories (n = 7 for each instrument). First, method transfer was performed to assess the separation quality and to set up the specific instrument parameters of Agilent® and Shimadzu® instruments. Second, the inter-laboratory study was performed following a protocol defined by the sending lab. Analytical results were examined regarding consistencies within- and between-laboratories criteria. Afterwards, the method reproducibility was estimated taking into account variances in replicates, between-days and between-laboratories. Reproducibility variance was larger than that observed during the first study involving only one single type of instrumentation. Indeed, we clearly observed an ‘instrument type’ effect. Moreover, the reproducibility variance was larger when considering all instruments than each type separately which can be attributed to the variability induced by the instrument configuration. Nevertheless, repeatability and reproducibility variances were found to be similar than those described for LC methods; i.e. reproducibility as %RSD was around 15 %. These results highlighted the robustness and the power of modern analytical SFC technologies to deliver accurate results for pharmaceutical quality control analysis.

Advancing stereoisomeric separation of an atropisomeric Bruton's tyrosine kinase inhibitor by using sub-2µm immobilized polysaccharide-based chiral columns in supercritical fluid chromatography.

BMS-986142 is a Bruton's tyrosine kinase inhibitor under development to treat several disease types. The compound contains three chiral elements: one chiral center and two chiral axes, resulting in three potential atropisomeric impurities in its drug substance and drug products. Separation of BMS-986142 atropisomers has been successfully achieved on an achiral polar-embedded C18 column in reversed-phase liquid chromatography (RPLC) and on polysaccharide-based chiral columns in RPLC and supercritical fluid chromatography (SFC). Compared to the RPLC chiral separation, the SFC atropisomeric separation on a sub-2 μm immobilized cellulose-based column is much more efficient and environmentally friendly. The analysis time in SFC was reduced by 8-fold compared to that in RPLC, and the method sensitivity in SFC on the sub-2 μm chiral column in 3.0mm I.D. was 2 to 4-fold better than that on 3 μm chiral columns in 4.6mm I.D.. Furthermore, our study suggests that the contribution to band broadening from the extra column volume (ECV) of modern commercial SFC instrument was not negligible for a 3.0mm I.D.×100mm column packed with 1.6 μm particles. This result reaffirms that there is a great need for further improvement of SFC instrument design in order to realize the full theoretical efficiency of both sub-2 μm achiral and chiral columns.

Unexpected carbonate salt formation during isolation of an enantiopure intermediate by supercritical fluid chromatography

The enantiomers of a chiral building block to be used in pre-clinical manufacturing were separated using supercritical fluid chromatography (SFC). Despite an extensive evaluation of different columns and solvent combinations followed by a careful optimization of the chromatographic method, the preparative separation suffered from low throughput and high solvent consumption. Consequently, additional improvements were necessary. By utilizing stacked injections, the chromatographic run time was almost halved, and the high solvent consumption was reduced by recycling of the two mobile phase components, carbon dioxide and methanol. The carbon dioxide was reprocessed by the SFC instrument, whereas methanol was evaporated and recycled from the fractions collected. Hence, the originally inefficient separation method was turned into a more sustainable one, and the desired enantiopure intermediate was delivered to be used in the following synthesis of the selected candidate drug. Unfortunately, when the intermediate was used in the subsequent amide coupling, a surprisingly poor yield was obtained. This was caused by an unexpected formation of a stable carbonate salt of the intermediate under the chromatographic conditions used. By removal of the carbonate prior to the amide coupling reaction, the manufacturing campaign could be saved, and the candidate drug was successfully delivered in time.

Enhanced fluidity liquid chromatography: A guide to scaling up from analytical to preparative separations

The evolution of supercritical fluid chromatography (SFC) instrumentation, improved detection capability, and expanded modifier range has led to extending the reach of SFC to the analysis of a broader spectrum of analytes beyond enantioselective separations. However, preparative SFC has yet to see the same technological revitalization, especially in regards to the purification of highly polar analytes. Enhanced fluidity liquid chromatography (EFLC) has been demonstrated as one of the ways to extend the applicable range of SFC instrumentation to highly polar analytes such as proteins, carbohydrates, and nucleotides. Despite recent applications of EFLC for challenging mixtures of hydrophilic metabolites and analogs, its viability in preparative purification, which is of great importance to the pharmaceutical industry, remains unknown. Herein, multiple chromatographic parameters that are critical to achieve feasible EFLC purification methods were investigated, including system pressure as a function of modifier composition (for several MeOH:H2O ratios), effect of diluent injection conditions on peak shape, and optimization of mass load with diluent composition. The usage of 50% acetonitrile or methanol diluents provided the most volumetric loading capacity. In the case of sucrose, leveraging higher analyte solubility in water proved to be more favorable than the volumetric loading capacity of diluents with higher organic content. In fact, an 80 mg injection of sucrose was possible on a 2 cm preparative HILIC column with minimal peak shape degradation. The combined information led to the successful demonstration of EFLC for the preparative separation of sugars using readily available MS-directed SFC instrumentation.

Kinetic performance of a 50 mm long 1.8 μm chiral column in supercritical fluid chromatography

Reduced plate heights (hr) of <2 were observed for the first time during the chiral separation of enantiomers, on sub-2μm particles with supercritical fluid chromatography (SFC). The enantiomers of trans-stilbene oxide, were separated on a 4.6×50mm, 1.8μm R,R-Whelk-O1 column, with hr as low as 1.93. The plumbing of a commercial SFC instrument was modified to create a low dispersion version. Without the modification performance was considerably worse.vanDeemter like plots of reduced plate height vs. flow rate, for trans-stilbene oxide, indicate that the optimum flow varied with% modifier. On a 4.6×250mm, 5μm R,R- Whelk-O1 column, the optimum flow was >4mL/min for 5% methanol in CO2, decreasing to <2mL/min for 40% methanol (more than a factor of 2). For a 4.6×50mm column packed with 1.8μm particles the optimum appeared to be near, or >5mL/min with 2.5%, 5%, and 10% methanol, decreasing to between 3 and 3.5mL/min at 40% methanol. This is the first time such shifts have been characterized. Since the solutes were the same in all cases, the differences are likely due to changes in solute diffusion coefficients caused by changes in modifier concentration, and pressure.Pump pressure requirements sometimes exceeded 500bar. It is shown that a 5mL/min flow rate is inadequate for use with 1.8μm particles in a 4.6mm ID column format. Instead, it is suggested to decrease the ID of the column to 3mm, where the optimum flow rates are on the order of 2mL/min with decreased tubing variance. Nevertheless, a number of sub-1min chromatograms are presented.

Supercritical fluid chromatography for GMP analysis in support of pharmaceutical development and manufacturing activities

Supercritical fluid chromatography (SFC) has long been a preferred method for enantiopurity analysis in support of pharmaceutical discovery and development, but implementation of the technique in regulated GMP laboratories has been somewhat slow, owing to limitations in instrument sensitivity, reproducibility, accuracy and robustness. In recent years, commercialization of next generation analytical SFC instrumentation has addressed previous shortcomings, making the technique better suited for GMP analysis. In this study we investigate the use of modern SFC for enantiopurity analysis of several pharmaceutical intermediates and compare the results with the conventional HPLC approaches historically used for analysis in a GMP setting. The findings clearly illustrate that modern SFC now exhibits improved precision, reproducibility, accuracy and robustness; also providing superior resolution and peak capacity compared to HPLC. Based on these findings, the use of modern chiral SFC is recommended for GMP studies of stereochemistry in pharmaceutical development and manufacturing.

Temperature effects in supercritical fluid chromatography: A trade-off between viscous heating and decompression cooling

The study of radial and axial temperature profiles always has been an area interest both in liquid chromatography (LC) and supercritical fluid chromatography (SFC). Whereas in LC always an increase in temperature is observed due to the dominance of viscous heating, in SFC, especially for low modifier content, a decrease in temperature is found due to the much larger decompression cooling. However, for higher modifier content and higher operating pressure, the temperature effects become a trade-off between viscous heating and decompression cooling, since in SFC the latter is a strong function of operating pressure and mobile phase composition. At a temperature of 40°C and for neat CO2, the effect of decompression cooling and viscous heating cancel each other out at a pressure 450bar. This pressure decreases almost linearly with volume fraction of methanol to 150bar at 25vol%. As a result, both cooling and heating effects can be observed when operating at high back pressure, large column pressure drops or high modifier content. For example at a back pressure of 150bar and a column pressure drop of 270bar decompression cooling is observed throughout the column. However at 300bar back pressure and the same pressure drop, the mobile phase heats up in the first part of the column due to viscous heating and then cools in the second part due to decompression cooling. When coupling columns (2.1mm×150mm, 1.8μm fully porous particles) at very high operating pressure (e.g. 750bar for 8vol%), the situation is even more complex. E.g. at a back pressure of 150bar and using 8vol% methanol, viscous heating is only observed in the first column whereas only decompression cooling in the second. Further increasing the inlet pressure up to 1050bar resulted in no excessive temperature differences along the column. This implies that the inlet pressure of SFC instrumentation could be expanded above 600bar without additional band broadening caused by excessive radial temperature differences.

Accurate measurements of frontal analysis for the determination of adsorption isotherms in supercritical fluid chromatography

The implementation of the traditional FA method is difficult with classical supercritical fluid chromatography (SFC) instruments. The instrument mixer and other sources of extra-column volumes are large and significantly broaden the fronts of injected plugs, which diminishes the precision and accuracy of the FA method. An SFC instrument was modified to permit more accurate determinations of adsorption isotherm data. The sample, the modifier, and CO2 are separately pumped via small volume connection tubes into a small volume mixer (250μL), where they are mixed into a homogeneous fluid fed to the column. The extra-column volumes and the column hold-up volume were accurately measured at each back pressure from the retention times of tracers. This modified instrument was used to measure the adsorption isotherm of S-naproxen by frontal analysis (FA) on a (R, R)-Whelk-O1 column, using a mixture of methanol (20%, v/v) and CO2 as the mobile phase. Its performance is studied at several different back pressures from 100 to 210bar. In all the experiments, the total flow rate was kept to a low value (1mL/min) in order to minimize the variation of the equilibrium constant along the column. Although a suitable breakthrough curve could not be obtained at low back pressures (<150bar) due to the closeness to the critical point pressure of the methanol/CO2 mixture, excellent results were obtained at higher back pressures (>150bar), conditions remote from the critical point and breakthrough curves with very sharp front shocks are obtained. The RSDs of the profiles recorded at each back pressures are excellent, better than 1%.

Review: Isomer Separation, Chiral Resolution, and Structure Elucidation Analyses Are the Future of Analytical Supercritical Fluid Chromatography—Mass Spectrometry

In 1822, almost 200 years ago, Baron Charles Cagniard de Latour (1777-1859) discovered the critical phenomena or critical state [1] [2]. At that time, it was described as a curiosity. However, it is now used as an advanced extraction and analytical technology in industry and research. Extraction with supercritical fluids (SFs) was welcome from the very beginning. In the present paper, the application to chromatography of the critical phenomena of compressed gases or overheated liquids as supercritical fluids will be discussed. Supercritical Fluid Chromatography (SFC) received an uneasy welcome, mainly because of the lack of commercially available analytical instruments. Even today, only a small number of industrial companies are manufacturing SFC instrumentation. A very small spectrum of columns is manufactured specially for SFC. SFC is not accepted by Pharmacopeia committees for the analysis of drugs and medical forms to the same extent as Gas Chromatography, High-Performance Liquid Chromatography or even Thin Layer Chromatography. The present status and future perspectives for SFC will be discussed.

Maximizing kinetic performance in supercritical fluid chromatography using state-of-the-art instruments

Recently, there has been a renewed interest in supercritical fluid chromatography (SFC), due to the introduction of state-of-the-art instruments and dedicated columns packed with small particles. However, the achievable kinetic performance and practical possibilities of such modern SFC instruments and columns has not been described in details until now. The goal of the present contribution was to provide some information about the optimal column dimensions (i.e. length, diameter and particle size) suitable for such state-of the-art systems, with respect to extra-column band broadening and system upper pressure limit. In addition, the reliability of the kinetic plot methodology, successfully applied in RPLC, was also evaluated under SFC conditions. Taking into account the system variance, measured at ∼85μL2, on modern SFC instruments, a column of 3mm I.D. was ideally suited for the current technology, as the loss in efficiency remained reasonable (i.e. less than 10% decrease for k>6). Conversely, these systems struggle with 2.1mm I.D. columns (55% loss in N for k=5). Regarding particle size, columns packed with 5μm particles provided unexpectedly high minimum reduced plate height values (hmin=3.0–3.4), while the 3.5 and 1.7μm packing provided lower reduced plate heights hmin=2.2–2.4 and hmin=2.7–3.2, respectively. Considering the system upper pressure limit, it appears that columns packed with 1.7μm particles give the lowest analysis time for efficiencies up to 40,000–60,000 plates, if the mobile phase composition is in the range of 2–19% MeOH. The 3.5μm particles were attractive for higher efficiencies, particularly when the modifier percentage was above 20%, while 5μm was never kinetically relevant with modern SFC instruments, due to an obvious limitation in terms of upper flow rate value. The present work also confirms that the kinetic plot methodology could be successfully applied to SFC, without the need for isopycnic measurements, as the difference in plate count between predicted and experimental values obtained by coupling several columns in series (up to 400mm) was on average equal to 3–6% and with a maximum of 13%.

SFC Instrument Modification to Allow Greater Flexibility in Method Development by Generating Mixtures of Solvents and Modifiers On-Line for Mobile Phase B Optimization

A binary supercritical fluid chromatography (SFC) system was further modified by the addition of a quaternary HPLC pump. The flow outlet of this pump was connected to the flow inlet of the existing modifier pump (“pump B”) by way of a custom-designed reservoir in such a way that blends of methanol with up to three additional solvents and/or additives could be delivered to pump B on-the-fly. Small amounts of modifiers (0.1–5 %) are typically mixed with mobile phase B in SFC to improve either the peak shape or to optimize the selectivity of the separation. As the critical influence of these modifiers on the separation of complex mixtures cannot a priori be predicted, incremental changes in mobile phase B compositions typically have to be evaluated on a trial-and-error basis. The instrumental modifications described in this paper eliminate the need to prepare these combinations off-line and the concomitant time requirement to re-purge supply and de-gasser solvent lines with each change in composition. By eliminating these steps, a significantly decrease in the overall SFC method development time can be achieved. Furthermore, such an on-line arrangement allows for greater flexibility in method development, that is, in the “fine-tuning” of the method once the stationary phase and gradient conditions have been established.

Evaluation of mobile phase gradient supercritical fluid chromatography for impurity profiling of pharmaceutical compounds

The use of gradient supercritical fluid chromatography (SFC) for the impurity profiling of pharmaceutical products is not widely practiced. Historically, the limited advancement in SFC instrumentation and the lag in column development have resulted in marginal sensitivity, selectivity and reproducibility when compared with high performance liquid chromatography (HPLC). Using a recently developed commercial module, which allows an ordinary HPLC to be converted to a SFC system, a significant improvement in sensitivity (up to ~12-fold) has been obtained over previous studies. This has allowed for the first time a "real-world" head-to-head comparison of SFC to HPLC for impurity profiling of pharmaceutical products in a regulated environment. Retention time reproducibility and low level impurity detection were found to be comparable to reversed phase liquid chromatography (RPLC), that is, single digit %relative standard deviations (RSDs) were obtained for impurities present at less than 0.1 area%. Furthermore, these results were obtained with drug loading levels (≤2 mg/mL) that are not only comparable to those employed with HPLC, but are dictated by the limited solubility of many drug candidates. The elution of impurities was generally found to be orthogonal to that obtained with RPLC, but it was still challenging to find SFC conditions that would separate all of the components in the mixtures studied. In terms of enhancing selectivity, small amounts of mobile phase additives (0.1-1%) and temperature optimization were found to have a greater impact in SFC method development versus RPLC. However, unlike gradient RPLC, the relative changes in baseline noise and slope were found to be a complex function of the experimental conditions, with the largest differences in noise levels being generally observed for the widest and steepest gradients. It is likely that this gradient related noise is more apparent now because other sources of noise in SFC have been reduced significantly.

Screening Strategy for Chiral and Achiral Separations in Supercritical Fluid Chromatography Mode

Supercritical fluid (SF) was discovered 200 years ago, but the use of this fluid as a mobile phase in chromatography only became popular fifty years ago. The development of the supercritical fluid chromatography (SFC) was progressing slowly due to technological problems since ten years; the interest for this chromatographic mode has been growing up as the construction of the SFC instruments is more or less similar with HPLC instruments. The main difference in SFC is the installation of a back pressure regulator which is implemented to control the pressure above the critical pressure. SFC is widely used in chiral chromatography where Polysaccharide phases are the most versatile in use. The mobile phase is mainly composed by CO(2) but the polarity can be increased by adding alcohol. The nature of the alcohol can change drastically the selectivity. The choice of the best tandem stationary phase / mobile phase is difficult to predict. Hence a full screening with different stationary phases and mobile phase solvents is often mandatory. For the achiral separation, SFC is more and more used. Achiral SFC can be classified as normal phase mode, it means that stationary phases are more polar than mobile phase and retention times decrease as polarity of the mobile phase increases. Most popular stationary phases are silica linked with polar group such as aminopropyl, cyanoprpyl, diol or 2-ethylpyridine. Mobile phase are generally composed by CO(2) and methanol. SFC can be used as a complementary technique for reversed phase HPLC or sometimes even to replace HPLC.

Solving multicomponent chiral separation challenges using a new SFC tandem column screening tool

A tool for improved tandem column chiral supercritical fluid chromatography (SFC) method development screening was prepared by modification of a commercial analytical SFC instrument with two different software-controllable, six position high-pressure column selection valves, each controlling a bank of five different columns and a pass through line. The resulting instrument, which has the ability to screen 10 different individual columns and 25 different tandem column arrangements, is a useful tool for facilitating the screening of tandem column SFC arrangements for separation of complex mixtures of stereoisomers or other multicomponent mixtures. Strategies for optimal use of the instrument are discussed, and several examples of the use of the instrument in developing tandem SFC methods for resolution of multicomponent mixtures are presented.

Fluidos supercríticos em química analítica. II. Cromatografia com fluido supercrítico: instrumentação

The first paper in this series discussed the basic theory involved in supercritical fluid chromatography (SFC) and how the technique progressed from gas and liquid chromatography. The first SFC instruments were simple adaptations of the commercially available liquid chromatographs with packed columns followed by modifications in gas chromatographs using open tubular capillary columns. In this paper, the most important aspects regarding instrumentation are covered, including practical, simple, and the most important, inexpensive solutions to build a home-made SFC system.

Direct injection of aqueous samples in packed column supercritical fluid chromatography of isosorbide-5-mononitrate from drug release testing

A method for the analysis of aqueous samples of isosorbide-5-mononitrate (5-ISMN) is presented. It is based on packed column supercritical fluid chromatography (SFC) using 20% of 2-propanol in carbon dioxide as the mobile phase and a diol silica column as the stationary phase. Using the described conditions it is possible to quantitate 5-ISMN released from Imdur® tablets in gastric media. The precision upon repeated injections was 2% (RSD) at the 20 μg/ml level ( n=8), using peak height measurements, when the solution was circulated through the sample loop of the injector. Samples from drug release testing that had been analyzed with reversed phase LC were analyzed with the present method and the results agreed well. It is also possible to monitor the drug released in a dissolution-testing vessel through direct on-line continuous loading (recirculation) of the sample loop of the SFC instrument.

Industrial assembly

Recently, there has been a renewed interest in supercritical fluid chromatography (SFC), due to the introduction of state-of-the-art instruments and dedicated columns packed with small particles. However, the achievable kinetic performance and practical possibilities of such modern SFC instruments and columns has not been described in details until now. The goal of the present contribution was to provide some information about the optimal column dimensions (i.e. length, diameter and particle size) suitable for such state-of the-art systems, with respect to extra-column band broadening and system upper pressure limit. In addition, the reliability of the kinetic plot methodology, successfully applied in RPLC, was also evaluated under SFC conditions. Taking into account the system variance, measured at ∼85 μL2, on modern SFC instruments, a column of 3 mm I.D. was ideally suited for the current technology, as the loss in efficiency remained reasonable (i.e. less than 10% decrease for k > 6). Conversely, these systems struggle with 2.1 mm I.D. columns (55% loss in N for k = 5). Regarding particle size, columns packed with 5 μm particles provided unexpectedly high minimum reduced plate height values (hmin = 3.0–3.4), while the 3.5 and 1.7 μm packing provided lower reduced plate heights hmin = 2.2–2.4 and hmin = 2.7–3.2, respectively. Considering the system upper pressure limit, it appears that columns packed with 1.7 μm particles give the lowest analysis time for efficiencies up to 40,000–60,000 plates, if the mobile phase composition is in the range of 2–19% MeOH. The 3.5 μm particles were attractive for higher efficiencies, particularly when the modifier percentage was above 20%, while 5 μm was never kinetically relevant with modern SFC instruments, due to an obvious limitation in terms of upper flow rate value. The present work also confirms that the kinetic plot methodology could be successfully applied to SFC, without the need for isopycnic measurements, as the difference in plate count between predicted and experimental values obtained by coupling several columns in series (up to 400 mm) was on average equal to 3–6% and with a maximum of 13%.