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.

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.

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.

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.

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.

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 CO-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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Use of the isopycnic plots in designing operations of supercritical fluid chromatography. V. Pressure and density drops using mixtures of carbon dioxide and methanol as the mobile phase.

The drops of pressure and density along chromatographic columns of different characteristics, eluted with different mixtures of carbon dioxide and methanol was mapped as functions of the column outlet pressure and the operating temperature. This paper extends an earlier report reporting the extent of the pressure and density drops along chromatographic columns eluted with neat CO(2)[1]. It illustrates the similarities and differences in the pressure and density profiles along columns operated with mixed mobile phases and with neat CO(2).

Pressure, temperature and density drops along supercritical fluid chromatography columns. II. Theoretical simulation for neat carbon dioxide and columns packed with 3-μm particles.

When chromatography is carried out with high-density carbon dioxide as the mobile phase, the required pressure gradient along the column is moderate but this mobile phase is highly compressible so, under certain experimental conditions, its density may decrease significantly along the column. Such an expansion absorbs heat and causes cooling of the column. The resulting heat transfer causes the formation of axial and radial gradients of temperature and density that may become large under certain conditions.

Pressure, temperature and density drops along supercritical fluid chromatography columns. I. Experimental results for neat carbon dioxide and columns packed with 3- and 5-micron particles.

The pressure drop and temperature drop on columns packed with 3- and 5-micron particles were measured using neat CO(2) at a flow rate of 5 mL/min, at temperatures from 20°C to 100°C, and outlet pressures from 80 to 300 bar. 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.

Use of the isopycnic plots in designing operations of supercritical fluid chromatography: IV. Pressure and density drops along columns.

The pressure- and the density-drops along a chromatographic column eluted with supercritical fluid carbon dioxide were mapped as a function of the outlet column pressure and the temperature on the P-T diagram of neat CO(2). At low densities, the viscosity of CO(2) is low, which is expected to result into a low pressure drop along the column. However, at these low densities, the volumetric flow rates of the mobile phase at constant mass flow rates are high, which might result into a high pressure drop along the column.

Unexpected retention behavior of supercritical fluid chromatography at the low density near critical region of carbon dioxide.

The retention factors of octylbenzene, octadecene, anthracene, and pyrene eluted from columns packed with neat silica and C(18)-bonded silica by pure carbon dioxide near its critical region increase with increasing temperature along low-density isopycnic lines. This behavior is markedly different from the one observed in nearby regions of the pressure-temperature diagram of CO(2), where the retention factors of these compounds decrease with increasing temperature along high-density isopycnic lines. Several possible reasons that could explain this reversal in the behavior of the retention factors are discussed.