Additives in chiral packed column super/subcritical fluid chromatography: A little goes a long way.

The introduction of additives has revolutionized super/subcritical fluid chromatography (SFC) by expanding the range of compounds that could be analyzed by the technique. From being considered a mere extension of gas chromatography, incorporation of a modifier, and subsequently an additive has made SFC a workhorse for chiral separations. Additives are by far the smallest component of the SFC mobile phase but can result in changing the polarity and acidity of the mobile phase, suppression of ionization, deactivation of the stationary phase, and act as an ion pairing agent. A wide variety of compounds have been tested as additives including but not limited to diethylamine, trifluoroacetic acid, ammonium acetate, and water. This review summarizes the different role played by additives in the SFC mobile phase. Further this work aims to help the reader by critically evaluating different additives used in enantiomeric separations with packed column SFC.

Application of retention modeling in chiral method development. I. Selection of isocratic composition for preparative separation with SFC.

Preparative chiral separations are carried out using chiral stationary-phases (CSP) employing isocratic composition mode to take advantage of stacking multiple injections within a single continuous operation. Development of the separation method, however, is not conducted directly in the preparative systems. Chromatographic systems at analytical scale are set up to screen multiple CSPs with various mobile-phases (MP) to detect a suitable CSP-MP combination. For faster method screening, solvent-gradients are implemented - operating from low to higher modifier composition, e.g. 5 to 70%. Once the right CSP-MP pair is detected, the isocratic method for preparative separation is developed through further experimental trials in the analytical system. The scope of the trial steps is generally limited to detecting a "good-enough" separation condition through one or two isocratic experiments. Ideally, the analyst should scout all possible isocratic conditions to detect the most suitable method; which, however, is not possible in high-throughput separation laboratories. In this report we demonstrate the utility of a simple set of algebraic equations, supported by an experimental protocol, in generating complete isocratic method options based on minimum number of experimental trials. The approach presented here was developed for chiral separation with supercritical-fluid chromatography. We also suggest an approach to identify an isocratic composition for the purification step. The process proposed in this report should be useful in developing better preparative separation methods in high-throughput laboratories.

Chiral chromatography method screening strategies: Past, present and future.

Method screening is an integral part of chromatographic method development for the separation of racemates. Due to the highly complex retention mechanism of a chiral stationary-phase, it is often difficult, if not impossible, to device predefined method-development steps that can be successfully applied to a wide group of molecules. The standard approach is to evaluate or screen a series of stationary and mobile-phase combinations to increase the chances of detecting a suitable separation condition. Such a process is often the rate-limiting step for high-throughput analyses and purification workflows. To address the problem, several solutions and strategies have been proposed over the years for reduction of net method-screening time. Some of the strategies have been adopted in practice while others remained confined in the literature. The main objective of this review is to revisit, critically discuss and compile the solutions published over the last two decades. We expect that making the diverse set of solutions available in a single document will help assessing the adequacy of existing screening protocols in laboratories conducting chiral separation.

Estimation of optimum injection volume during preparative chiral separation.

Estimation of injection volume during a preparative chiral separation can be challenging. Commonly one attempts to maximize the injection volume to reduce total separation time. The factors that limit increasing injection volume are (a) purity constraint(e.g. enantiomeric excess) and (b) criterion of product recovery. Standard industrial practice is to successively inject increasing volume of sample mixture until two adjoining peaks (e.g. peaks of two enantiomers) touch respective baselines. Separation scientists may spend considerable time and material to detect this injection volume before starting the stack-injection run. This increased method-development time increases time spent on the instrument, resulting in decreased efficiency. In this report we demonstrate the utility of a mathematical model-based approach that can be employed for faster estimation of this optimum injection volume. Note that the model does not intend to detect the theoretical maximum injection volume, rather tries to mimic the experimental optimization approach through simulation. The method requires experimental data from one initial trial run as input to predict the final "optimum" injection volume, thus saving time and material compared to situations that require multiple trial runs. The proposed model is simple enough to be implemented in a Microsoft Excel spreadsheet, but offers reasonably accurate estimation. Although the example presented in this report is of preparative separation of a racemic mixture with supercritcal fluid chromatography (SFC), in general the model is applicable to any preparative separation under isocratic conditions where the chromatographic peak follows Langmuir adsorption isotherm behavior. A description of the fundamental basis of the model is presented here along with experimental results that demonstrate its utility.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

Accurate on-line mass flow measurements in supercritical fluid chromatography.

This work demonstrates the possible advantages and the challenges of accurate on-line measurements of the CO2 mass flow rate during supercritical fluid chromatography (SFC) operations. Only the mass flow rate is constant along the column in SFC. The volume flow rate is not. The critical importance of accurate measurements of mass flow rates for the achievement of reproducible data and the serious difficulties encountered in supercritical fluid chromatography for its assessment were discussed earlier based on the physical properties of carbon dioxide. In this report, we experimentally demonstrate the problems encountered when performing mass flow rate measurements and the gain that can possibly be achieved by acquiring reproducible data using a Coriolis flow meter. The results obtained show how the use of a highly accurate mass flow meter permits, besides the determination of accurate values of the mass flow rate, a systematic, constant diagnosis of the correct operation of the instrument and the monitoring of the condition of the carbon dioxide pump.

Determination of the adsorption isotherm of the naproxen enantiomers on (S,S)-Whelk-O1 in supercritical fluid chromatography.

The adsorption isotherms of the naproxen enantiomers were measured on a Kromasil Whelk-O1 column, eluted with mixtures of supercritical carbon dioxide and methanol or ethanol. Five chromatographic methods were used: frontal analysis, frontal analysis by characteristic points, elution by characteristic points, retention time method and the inverse method. In all methods, the effects of the two modifiers were compared. The use of these methods suffers from limitations due to supercritical fluid chromatographic instruments. These problems and drawbacks are discussed. The accurate and precise determination of the isotherm parameters was not possible with the instrument currently used. In contrast, the different methods allow to show qualitatively that the adsorption of the naproxen enantiomers show heterogeneous mechanism on the adsorbent surface common in chiral chromatography. Finally, the experimental high concentration elution band profiles of naproxen are compared with those calculated from the isotherm parameters provided by the five different methods.

Distributed pore model for bio-molecule chromatography.

One of the main peculiarities in protein chromatography is that the adsorbing proteins and the adsorbent pores have comparable sizes. This has the consequence that the pore accessibility depends not only on the solute size but also on the loading conditions of the adsorbent because protein adsorption significantly reduces the size of the pores. A model that accounts for the pore size distribution of the stationary phase and for the pore shrinkage due to protein adsorption has been developed to describe mass transport and adsorption in the porous particles. This model has been shown to be equivalent to the general rate model (GRM) in the case of processes under highly diluted conditions with little adsorption. This implies that the model parameters determination follows the same procedure as for the classical GRM. The new pore model has been applied and compared to the GRM for the simulation of lysozyme breakthrough experiments and for the prediction of 5% dynamic binding capacity values solely based on static capacity measurements.

Interpretation of dynamic frontal analysis data in solid/supercritical fluid adsorption systems. I: theory.

A theory is proposed to relate the elution times of the adsorption front shocks of breakthrough curves recorded during classical dynamic frontal analysis (FA) experiments with selected compounds and their adsorption isotherms in solid/supercritical fluid adsorption systems. The actual density and viscosity of binary mixtures of CO2 and methanol were obtained from the NIST REPPROP software. Diluted solutions of S-naproxen were considered (<2% in mass) but the possible effects of the analyte concentration on the viscosity and the density of the eluent percolating through the column were neglected. This allows the determination of the excess adsorption isotherm (or Gibbs excess isotherm) of the adsorbed analyte in the whole column at constant mass and volumetric flow rate of pure CO2 and of the modifier solution. A local Langmuir adsorption isotherm and a constant saturation capacity were assumed in the calculations. The variation of the adsorption-desorption constant with the eluent density was taken from the experimental variation of the retention factor of S-naproxen on a chiral column packed with Whelk-O1 particles. The results show that the isotherm parameters obtained from the best adjustment of the Langmuir model to the SFC excess adsorption data deviates by less than 7% from the assumed saturation capacity and from the average of the equilibrium constant along the chromatographic column. In practice, this conclusion holds true provided that the precision of the measurement of elution times of front shocks of breakthrough curves is better than 1% and that the maximum surface coverage qexp,max/qS is at least equal to 20%.

Efficiency of supercritical fluid chromatography columns in different thermal environments.

The efficiency of a packed column eluted with supercritical carbon dioxide at 323K and outlet pressures from 90 to 150bar was studied with the column in two different thermal environments. The 150mm×2.0mm ID stainless steel column was packed with spherical 5-µm porous silica particles with a covalently bonded nonpolar stationary phase, and the test solutes were normal alkanes. When operated in a convective air bath the column exhibited severe efficiency losses when its outlet pressure was below 120bar. The efficiency of the same column enclosed in a shell made of foam insulation was restored at low outlet pressures down to 100bar. The van Deemter plots showed an abnormal dependence of the plate height (HETP) on the flow rate at low outlet pressures, exhibiting a maximum in the HETP at flow rates around 1mL/min and a 20-bar pressure drop. The large efficiency losses at low outlet pressures are due to radial temperature gradients associated with enthalpic expansion and cooling of the mobile phase. The separations were simulated by a numerical model that accounts for axial and radial gradients in the temperature and density along the column. The abnormal van Deemter plots arise from competing processes affecting the radial distribution of the solute migration velocity along the column. The negative impact on efficiency is greatest when the density profile of the mobile phase along the column is close to the critical isopycnic line. The efficiency improves at increased flow rates because of increased cooling at larger pressure drops and increased density along the entire length of the column. The model predicts the unusual trends in the van Deemter plots, but the calculated results at low outlet pressures are strongly influenced by small variations in the porosity distribution in the column, limiting the accuracy of the predicted HETP values. In spite of these difficulties, the model has enabled a detailed analysis of the effects of temperature, pressure and flow rate on the thermal properties of the mobile phase, and their impact on the radial distribution of the solute velocities along the column. This work provides a better appreciation of the factors that cause excess efficiency loss at low outlet pressures, a phenomenon that lacked a convincing explanation for over 40 years. Finally, a simplified form of the model, which ignores the radial gradients, provided accurate results only at the highest outlet pressure. Calculations done by the simplified model are much faster, and it can be recommended for simulation of SFC processes at sufficiently high outlet pressures.

Accurate measurements of experimental parameters in supercritical fluid chromatography. I. Extent of variations of the mass and volumetric flow rates.

Previous reports have highlighted the influence of the properties of the mobile phase flow rate on the column performance achieved in supercritical fluid chromatography (SFC). In SFC both the mass and the volumetric flow rates have unique influences on the chromatographic performance and the determination of their exact values is critical. It is well understood that the mass flow rate stays constant along an SFC system whereas the volumetric flow rate may vary considerably, but the extent of these variations and the role of the individual operating parameters in influencing these variations have not been clearly reported yet. The factors that control the mass and the volumetric flow rates in an SFC system are discussed and the possible extent of variations of these flow rates under different operating pressures and temperatures are demonstrate quantitatively.

Extended zones of operations in supercritical fluid chromatography.

The pressure and temperature ranges within which supercritical fluid chromatography is operated are generally decided based upon limitations imposed by the instrument or by the stationary phase. Because the maximum pump outlet pressure of most commercial instruments is near 400 bar and the maximum temperature at which most chiral stationary phases are stable is usually below 318 K, the possibility of performing analyses at sub-ambient temperatures (e.g., below 293 K) and under sub-critical pressures (i.e. below 73.8 bar) should be explored. This work investigates the performance of separations made in this unusual zone of operations, which might be attractive for some relevant SFC separations.