Abhijit Tarafder

Pressure, temperature and density drops along supercritical fluid chromatography columns in different thermal environments. III. Mixtures of carbon dioxide and methanol as the mobile phase.

The pressure, temperature and density drops along SFC columns eluted with a CO2/methanol mobile phase were measured and compared with theoretical values. For columns packed with 3- and 5-μm particles the pressure and temperature drops were measured using a mobile phase of 95% CO2 and 5% methanol at a flow rate of 5mL/min, at temperatures from 20 to 100°C, and outlet pressures from 80 to 300bar. The density drop was calculated based on the temperature and pressure at the column inlet and outlet. The columns were suspended in a circulating air bath, either bare or covered with foam insulation. The experimental measurements were compared to theoretical results obtained by numerical simulation. For the convective air condition at outlet pressures above 100bar the average difference between the experimental and calculated temperature drops and pressure drops were 0.1°C and 0.7% for the bare 3-μm column, respectively, and were 0.6°C and 4.1% for the insulated column. The observed temperature drops for the insulated columns are consistent with those predicted by the Joule-Thomson coefficients for isenthalpic expansion. The dependence of the temperature and the pressure drops on the Joule-Thomson coefficient and kinematic viscosity are described for carbon dioxide mobile phases containing up to 20% methanol.

A scaling rule in supercritical fluid chromatography. I. Theory for isocratic systems.

Scaling is regularly done in chromatography either to transfer a successfully designed method of analysis developed in one system to another system, or to scale-up a separation method developed in analytical scale to preparative scale. For liquid chromatography there are well-tested guidelines for scaling, which makes it a routine job. For supercritical fluid chromatography (SFC), on the other hand, neither do we have any well-understood principles behind scaling nor do we know how far the strategies applied in LC could be applicable to SFC. In this article, we have addressed these issues and proposed a rule applicable for scaling isocratic methods between different SFC systems and column dimensions under commonly used operating temperatures and pressures. We have shown that the scale-up and method transfer techniques used in LC can be applied to SFC, provided we ensure that both the original and the target systems in SFC operate at the same average density. The current article will present the theory, discuss the extents of applicability of this rule, and outline its limitations. In an accompanying article implementation of this rule in various practical situations will be presented.

A systematic investigation of sample diluents in modern supercritical fluid chromatography

This paper focuses on the possibility to inject large volumes (up to 10μL) in ultra-high performance supercritical fluid chromatography (UHPSFC) under generic gradient conditions. Several injection and method parameters have been individually evaluated (i.e. analyte concentration, injection volume, initial percentage of co-solvent in the gradient, nature of the weak needle wash solvent, nature of the sample diluent, nature of the column and of the analyte). The most critical parameters were further investigated using in a multivariate approach. The overall results suggested that several aprotic solvents including methyl tert-butyl ether (MTBE), dichloromethane, acetonitrile or cyclopentyl methyl ether (CPME) were well adapted for the injection of large volume in UHPSFC, while MeOH was generally the worst alternative. However, the nature of the stationary phase also had a strong impact and some of these diluents did not perform equally on each column. This was due to the existence of a competition in the adsorption of the analyte and the diluent on the stationary phase. This observation introduced the idea that the sample diluent should not only be chosen according to the analyte but also to the column chemistry to limit the interactions between the diluent and the ligands. Other important characteristics of the ideal SFC sample diluent were finally highlighted. Aprotic solvents with low viscosity are preferable to avoid strong solvent effects and viscous fingering, respectively. In the end, the authors suggest that the choice of the sample diluent should be part of the method development, as a function of the analyte and the selected stationary phase.

Estimations of temperature deviations in chromatographic columns using isenthalpic plots. I. Theory for isocratic systems.

We propose to use constant enthalpy or isenthalpic diagrams as a tool to estimate the extent of the temperature variations caused by the mobile phase pressure drop along a chromatographic column, e.g. of its cooling in supercritical fluid and its heating in ultra-performance liquid chromatography. Temperature strongly affects chromatographic phenomena. Any of its variations inside the column, whether intended or not, can lead to significant changes in separation performance. Although instruments use column ovens in order to keep constant the column temperature, operating conditions leading to a high pressure drop may cause significant variations of the column temperature, both in the axial and the radial directions, from the set value. Different ways of measuring these temperature variations are available but they are too inconvenient to be employed in many practical situations. In contrast, the thermodynamic plot-based method that we describe here can easily be used with only a ruler and a pencil. They should be helpful in developing methods or in analyzing results in analytical laboratories. Although the most effective application area for this approach should be SFC (supercritical fluid chromatography), it can be applied to any chromatographic conditions in which temperature variations take place along the column due to the pressure drop, e.g. in ultra-high pressure liquid chromatography (UHPLC). The method proposed here is applicable to isocractic conditions only.

Use of isopycnic plots to understand the role of density in SFC – I. Effect of pressure variation on retention factors

This paper aims to demonstrate the effect of pressure variations in modifying analyte retention behavior in SFC. There is a general understanding that in SFC increasing pressure decreases the retention factor (k), and vice versa. What is not clearly discussed or explained in any recent literature is that these variations can be very different at different operating pressures, temperatures and modifier concentrations. It is important to have a clearer understanding on these variabilities during method development and results analysis. In this paper the nature of k variation with pressure, at different temperatures and modifier concentrations, will be explained with the help of isopycnic plots of CO2 and CO2+methanol mixtures.

Scaling rule in SFC. II. A practical rule for isocratic systems

Scaling methods, either from analytical to analytical systems or from analytical to preparative systems and vice versa, are commonly performed in chromatography. For liquid chromatography there exist geometric rules for scaling, which provide guidelines to select column dimensions, particle sizes and flow rates. For SFC, on the other hand, there are no such rules or any well-understood principles behind scaling. In a recent report [1] this issue was addressed, proposing a rule of maintaining the same average density in the target system as it was in the original system. The problem with the criterion of maintaining average density, however, is the availability of density data. Not only one needs access and relevant experience of working with physical property data, in many cases density data may not be available at all. The current report demonstrates that a simpler approach, of matching average pressures, is equally applicable for acceptable scaling over most of the operating conditions used in SFC.

Designs and methods for interfacing SFC with MS

Hyphenating SFC with MS is now routinely performed in analytical laboratories. Major instrument providers supply commercial solutions for coupling SFC and MS, which has facilitated wider adoption of the technology. The current status, however, could be achieved based on the work done by many researchers over decades. Interfacing SFC with MS posed some unique challenges, compared to interfacing MS with LC or GC, demanding special solutions. Several interface designs were tried and tested over the years before suitable solutions could be detected. Additional measures, such as (a) mixing SFC mobile-phase with an additional liquid solvent at the column outlet, and (b) heating the interfacing device, had to be adopted to address some specific challenges. Although such modifications and measures look diverse, there is one factor that drove most of them - compressibility of SFC mobile-phase. There are two objectives of this review - (1) to compile various insights which were reported on describing and optimizing SFC-MS interfacing processes, and (2) to link these insights with the fundamental issue of solvent compressibility.

First inter-laboratory study of a Supercritical Fluid Chromatography method for the determination of pharmaceutical impurities

Graphical abstract Figure. No Caption available. HighlightsInter‐laboratory study was conducted for the first time in SFC.This study involved 19 participating laboratories from 4 continents and 9 different countries.Results consistencies within‐ and between‐laboratories were deeply examined.The method reproducibility was estimated taking into account variances in replicates, between‐days and between‐laboratories.Repeatability and reproducibility variances were found to be similar or better than those described for LC methods. ABSTRACT Supercritical Fluid Chromatography (SFC) has known a strong regain of interest for the last 10 years, especially in the field of pharmaceutical analysis. Besides the development and validation of the SFC method in one individual laboratory, it is also important to demonstrate its applicability and transferability to various laboratories around the world. Therefore, an inter‐laboratory study was conducted and published for the first time in SFC, to assess method reproducibility, and evaluate whether this chromatographic technique could become a reference method for quality control (QC) laboratories. This study involved 19 participating laboratories from 4 continents and 9 different countries. It included 5 academic groups, 3 demonstration laboratories at analytical instrument companies, 10 pharmaceutical companies and 1 food company. In the initial analysis of the study results, consistencies within‐ and between‐laboratories were deeply examined. In the subsequent analysis, the method reproducibility was estimated taking into account variances in replicates, between‐days and between‐laboratories. The results obtained were compared with the literature values for liquid chromatography (LC) in the context of impurities determination. Repeatability and reproducibility variances were found to be similar or better than those described for LC methods, and highlighted the adequacy of the SFC method for QC analyses. The results demonstrated the excellent and robust quantitative performance of SFC. Consequently, this complementary technique is recognized on equal merit to other chromatographic techniques.

A study on the onset of turbulent conditions with supercritical fluid chromatography mobile-phases

Following a recent publication [1], the topic of turbulent flow in SFC has generated both interest and questions. Liquid-like density, coupled with significantly low viscosity of CO2-based mobile-phases may result in high Reynolds number (Re) - higher than what represents laminar flow conditions, reaching the so-called turbulent regions. Although such turbulent flows can form only in the connecting tubings, thus not directly affecting the chromatographic process, it is important to know under many situations, whether the flow inside the tubing is laminar or turbulent. In this report a comprehensive guideline to identify the possibilities of turbulent flow conditions is provided through a series of charts. Flow properties depend on state conditions (composition, pressure and temperature) and also on the tubing material and geometry. Here guidelines to detect the onset of turbulent conditions is provided for cylindrical stainless-steel tubings of different internal diameters (i.d.) under a wide range of SFC mobile-phase conditions.

Visualizing Events Under Solvent-Gradient and Insights Thereof

This paper describes graphical methods to visualize variations in mobile phase composition inside a chromatographic column under solvent-gradient conditions, and how these variations affect movement of analyte bands. Based on the visualization techniques, the report explains how variations in composition “experienced” by an analyte, which is moving, are different from variations experienced by a static point inside the column. For example, if a linear gradient is created by the system, every point inside the column will experience the same linear variation of composition, although at different times, assuming that the solvent-gradient created at the mixer propagates through the system intact and undisturbed. For an analyte, on the other hand, the variation could be strongly non-linear because normally it travels with varying speed along the column during a gradient run. And because different analytes travel with different speeds, solvent-gradient experienced by the analytes will not be the same, even under the same method condition. Although these events are captured in the fundamental equation of solvent-gradient chromatography and generally understood by researchers working with gradient theories, the visualization methods discussed here may provide a clearer imagery of events for wider appreciation.