Carbon Dioxide-based Mobile Phases Research Articles

Supercritical fluid chromatography and liquid chromatography for isomeric separation of a multiple chiral centers analyte

The development of a chiral separation strategy has always been a challenge of crucial importance, particularly in the pharmaceutical field. Chromatographic methods have become popular, particularly High Performance Liquid Chromatography and Supercritical Fluid Chromatography from a preparative scale point of view. A bioactive compound bearing three stereogenic centers was entrusted in our laboratory and the aim of this work was to obtain the complete resolution of the eight stereoisomers. Nine different polysaccharide-based columns were tested in SFC under various carbon dioxide-based mobile phases. The use of a single chiral column Lux Cellulose-2 under 30% 2-PrOH in carbon dioxide, at a flow-rate of 1 mL/min, column temperature of 40°C, 120 bar outlet pressure allowed the obtention of eight peaks. To further improve the resolution of the two last isomers, two columns were serially coupled . The results obtained with the six different combinations are discussed. The tandem column supercritical fluid chromatography has demonstrated to be a useful technique to resolve the eight stereoisomers on Lux Cellulose-2//Cellulose-2 tandem of coupled columns with 30% 2-PrOH in carbon dioxide, at a flow-rate of 1 mL/min, column temperature of 40°C and 120 bar outlet pressure, despite a long analysis time. In order to compare the two methods (i.e supercritical and liquid), chiral liquid chromatography under polar aqueous-organic mode, polar organic mode and normal-phase mode, was implemented. The last mode allowed the full baseline resolution of the eight isomers on Cellulose-5 CSP, with 20% 2-PrOH in n-heptane at a flow-rate of 0.8 mL/min, at 25°C, λ=220 nm. The limits of detection and of quantification were determined for this method and the best values obtained for isomer 8 were equal to 2.84 and 9.37 nM respectively. Finally, a small-scale preparative separation of the multiple chiral centers compound was implemented on Cellulose-5 CSP within 10% 2-PrOH in n-heptane in order to study the stereoisomer elution order on Cellulose-2, Cellulose-5 and Chiralpak AD-H, under EtOH or 2-PrOH in n-heptane mobile phases, and partial reversal elution orders were observed.

An attempt to estimate ionic interactions with phenyl and pentafluorophenyl stationary phases in supercritical fluid chromatography

Pentafluorophenyl-bonded silica (PFP) phases employed in high-performance liquid chromatography (HPLC) often provide very different results depending on the column manufacturer. PFP phases also provide significantly different selectivity from non-fluorinated aromatic phases. As all HPLC columns can also be employed with carbon dioxide-based mobile phases, PFP phases can also be useful to supercritical fluid chromatography (SFC). However, whether they provide adequate retention and selectivity in SFC conditions is necessary for them to find applicability with this technique.In our laboratory, a column classification for packed column SFC was designed, based on the solvation parameter model, which currently comprises data for about eighty different columns. In this paper, we present the characterization of eleven PFP phases provided by different manufacturers used with carbon dioxide–methanol mobile phases. The columns are compared to fifteen other non-fluorinated phenyl and diphenyl phases in terms of their retention and separation characteristics assessed by the solvation parameter model with five Abraham descriptors. The latter is insufficient for an accurate description of retention mechanisms on the PFP phases, thus two extra terms accounting for ionic interactions with anions and cations (D− and D+), previously developed for HPLC in hydrophilic interaction mode (HILIC), are introduced. While some approximations are necessary regarding the true pH and pKa values in CO2-based mobile phases, the retention models are significantly improved by this addition, allowing to integrate ionizable analytes in the test set for evaluation of ionic interactions in the chromatographic systems.

Separation of linear gramicidins using carbon dioxide-containing mobile phases

Packed-column supercritical-fluid chromatography (pSFC) is presented as a novel method for separating and analyzing gramicidin samples. By use of methanol-modified carbon dioxide as a mobile phase the pentadecapeptides gramicidin A (gA), gramicidin B (gB), and gramicidin C (gC) are readily separated and eluted from a PRP-1 poly(styrene-divinylbenzene) column. Although optimum separation conditions are typically achieved near a column temperature of 40 degrees C, a column pressure of 11 MPa, and 30% methanol modifier, pressure and modifier gradients around these values are also found to improve the overall separation time. Measurements indicate that the mobile phase solubility of gramicidin under these conditions is 5.0+/-0.4 microg mL(-1). Collection of individual peaks during chromatography achieved analytical-scale isolation of 2 microg refined gC from 20 microg injected gramicidin D. Further, supercritical-fluid extraction of 200 microg gramicidin D from a Chromosorb 102 support packed into the vessel produced 57 microg gA in 90% purity. The results establish that carbon dioxide-based mobile phases can be successfully used for the separation of individual gramicidin species.

Direct, preparative enantioselective chromatography of propranolol hydrochloride and thioridazine hydrochloride using carbon dioxide-based mobile phases

In this paper, we describe the direct, preparative enantioselective chromatography of racemic ( rac)-propranolol hydrochloride (HCl) and rac-thioridazine·HCl using Chiralpak AD chiral stationary phase and mobile phase systems containing carbon dioxide and methanol without the use of basic or acidic additives. Isolated fractions of propranolol·HCl were positively identified by mass spectrometry, Beilstein flame test, melting point, and chemical analysis to be HCl enantiomers of propranolol·HCl salts exhibited characteristic mass spectra peaks at 36 and 38 mass-to-charge ratio in the expected 3:1 isotopic ratio for the solute that were absent in the mass spectra for the free-base forms. To our knowledge, the direct, preparative enantioselective isolation of HCl enantiomeric salts of rac-propranolol and of rac-thioridazine have not been previously demonstrated and published.

Empirical relationship between chiral selectivity and mobile phase modifier properties

An empirical relationship was derived which relates properties of the mobile phase modifier to the chiral selectivity factor for a given analyte/chiral selector combination. Using carbon dioxide and heptane-based mobile phases, the effect of various mobile phase modifiers on Pirkle-type stationary phases may be accurately modeled using a two-parameter equation. Similar results are obtained using cellulosic stationary phases with carbon dioxide-based mobile phases. Modeling separations performed using heptane-based mobile phases with cellulosic stationary phases were not successful. The predictive ability of this modeling approach was demonstrated using novel modifiers and chiral analytes.

Comparison of 1,1,1,2-tetrafluoroethane and carbon dioxide-based mobile phases for packed column supercritical fluid chromatography

Highly polar, homogeneous mobile phases have a number of advantages over carbon dioxide-based mixed mobile phases for supercritical fluid chromatography. While these fluids have been demonstrated to have very high eluotropic strengths in capillary systems, their utility for packed columns lies in their ability to overcome the deleterious effects of residual silanol groups present on the bonded-phase surface. A particularly promising fluid, 1,1,1,2-tetrafluoroethane (HFC-134a), is characterized with respect to eluotropic strength, selectivity, efficiency and temperature effects. These results are compared to those obtained with carbon dioxide and methanol-modified carbon dioxide mobile phases on a silica column. HFC-134a displays different selectivities, efficiencies, eluotropic strength and temperature dependencies on retention than either of the two reference mobile phases.

Temperature effects on selectivity using carbon dioxide-based mobile phases on silica-based packed columns near the mixture critical point

The temperature dependence on selectivity using modified carbon dioxide mobile phases with silica-based packed columns is found to be primarily related to the hydrogen bonding character of the analytes. Selectivity between hydrogen bond acceptor compounds is enhanced at temperatures above the mixture critical temperature. Selectivity for these compounds is further enhanced using hydrogen bond donating modifiers, such as alcohols. Selectivity between hydrogen bond donor/acceptor compounds is enhanced at lower temperatures, below the mixture critical temperature, regardless of the modifier used. Analysis of the enthalpic contributions to retention indicate that the main driving force behind these variations in temperature and modifier effects are the differences in solute heat of adsorption on the silica stationary phase.

Optimization of chiral resolution using packed columns with carbon dioxide-based mobile phases

Optimization of chiral resolution, using carbon dioxide based mobile phases, must take into consideration the individual contributions of analyte retention, selectivity, and efficiency. Each of these factors may be independently affected by changes in pressure, temperature, or state of the mobile phase. The ability to control retention by different means reflects an advantage of carbon dioxide based mobile phases over conventional HPLC mobile phases. Utilization of this advantage requires that the effects of each of these factors on each contributor to resolution be known. The cumulative effect that each of these variables has on retention, selectivity and efficiency suggests that maximum resolution is obtained using low pressures and temperatures. Maximum resolution (at fixed k′) results from low temperatures and high pressures. The latter may be of more practical importance when speed of analyses and detection limits are considered. Chirality 9:672–677, 1997. © 1997 Wiley-Liss, Inc.

Temperature effects for chiral separations using various bulk fluids in near‐critical mobile phases

As supercritical fluid chromatography becomes more accepted as a facile means for the separation of chiral compounds, the need for mobile phases that can readily solubilize these polar compounds grows. Prior studies suggest that HFC-134a may prove suitable due to its very high eluotropic strength compared to carbon dioxide-based mobile phases. A comparison is made between ethanol-modified carbon dioxide, HFC-134a, and decafluoropentane as to their relative eluotropic strength, selectivity, and efficiency for three chiral compounds using a Whelk O-1 chiral bonded phase. The bulk component of the mobile phase was found to have relatively little effect on chiral selectivity over the range of 5° to 95°C. Chirality 9:693–698, 1997. © 1997 Wiley-Liss, Inc.

Temperature effects for chiral separations using various bulk fluids in near-critical mobile phases

As supercritical fluid chromatography becomes more accepted as a facile means for the separation of chiral compounds, the need for mobile phases that can readily solubilize these polar compounds grows. Prior studies suggest that HFC-134a may prove suitable due to its very high eluotropic strength compared to carbon dioxide-based mobile phases. A comparison is made between ethanol-modified carbon dioxide, HFC-134a, and decafluoropentane as to their relative eluotropic strength, selectivity, and efficiency for three chiral compounds using a Whelk O-1 chiral bonded phase. The bulk component of the mobile phase was found to have relatively little effect on chiral selectivity over the range of 5° to 95°C. Chirality 9:693–698, 1997. © 1997 Wiley-Liss, Inc.

Separation of trans/ cis- α- and β-carotenes by supercritical fluid chromatography : I. Effects of temperature, pressure and organic modifiers on the retention of carotenes

A partial separation of the isomers of α- and β-carotenes has previously been achieved by non-aqueous reversed-phase chromatography on octadecyl silica columns eluted with mixtures of hexane and polar modifiers. We show that improved results are obtained by the use of mobile phases based on liquid or supercritical carbon dioxide. Polar modifiers have similar effects on chromatographic selectivity in the non-aqueous reversed-phase and supercritical fluid chromatography systems, suggesting that the retention mechanism is the same in both cases (predominantly partition). Withthe carbon dioxide-based mobile phases, it was possible to optimize chromatographic efficiency by varying the temperature and pressure. The use of both binary and ternary mixtures was investigated.