Chiral stationary phases in concert with homologous chiral mobile phase additives: Push/pull model

A new “push/pull” model has been developed to explain synergistic effects observed in a system in which a chiral stationary phase (CSP) is used in combination with its homologous chiral mobile phase additive (CMPA). The model predicts the beneficial CMPA enantiomer and the counterproductive CMPA enantiomer a priori. Thus, an (R)-CSP will obtain positive synergism from a homologous (S)-CMPA and negative synergism from a homologous (R)-CMPA. The reverse is true for an (S)-CSP. The importance of structural homology between the CSP and the CMPA is demonstrated. Furthermore, the analysis time is decreased relative to the use of a CSP alone, because the retention time of the analyte peaks decreases when the CMPAs are used. © 1993 Wiley-Liss, Inc.     

Enantiomeric separation of amino alcohols using Z-L-glu-L-pro or Z-L-glu-D-pro as chiral counter ions and hypercarb as the solid phase

This paper describes the synthesis of two new N-derivatized dipeptides. The two compounds, N-benzyloxycarbonyl-L-glutamyl-L-proline (Z-L-glu-L-pro) and N-benzyloxycarbonyl-L-glutamyl-D-proline (Z-L-glu-D-pro), were tested as chiral counter ions for the enantiomeric resolution of amino alcohols. The chiral counter ions were dissolved in a polar solvent, e.g., methanol and porous graphitic carbon, Hypercarb, were used as the achiral solid phase. The enantiomers of several of the tested compounds were baseline separated using Z-L-glu-L-pro as the chiral counter ion but no enantioselective retention was obtained using its diastereoisomer Z-L-glu-D-pro. The influence of solute structure as well as the importance of converting the chiral counter ion to its dianionic form will be described together with the effect of column temperature on enantioselective retention. Chirality 9:650–655, 1997. © 1997 Wiley-Liss, Inc.     

Enantioseparation of chiral perfluorooctane sulfonate (PFOS) by supercritical fluid chromatography (SFC): Effects of the chromatographic conditions and separation mechanism

Perfluorooctane sulfonate (PFOS) is one of the most frequently detected perfluoroalkyl substances in environmental and human samples. Previous studies have shown that nonracemic PFOS in biological samples can be used as a marker of PFOS exposure sources. In recent years, supercritical fluid chromatography (SFC) has emerged as a powerful method to separate chiral compounds. In this study, a method of perfluoro‐1‐methylheptane sulfonate (1 m‐PFOS) enantioseparation by SFC was established. The optimal separation was obtained using a Chiralpak QN‐AX column with CO₂/2‐propanol (70/30, v/v) as the mobile phase with a flow rate of 1 mL/min, column temperature was 32°C, and BPR pressure was 1800 psi. The resolution (Rs) and retention time were 0.88 and 130 minutes, respectively. This method is more economic and greener than HPLC. Modifier pH and column temperature were determined to be significant factors of SFC chiral separation. Modifier pH is negatively correlated with the retention factors and Rs. Adsorption thermodynamics were used to explain the influence of temperature change, and it was concluded that the transfer of two enantiomers from the mobile phase to the stationary phase is enthalpy‐driven. Enantioseparation of 1 m‐PFOS by SFC follows the same rules of ion exchange as those for the chiral separation by HPLC.     

Enantioseparation of hydrochloride salts using carbon dioxide-based mobile phases with on-line polarimetric detection

Most HPLC enantioseparations of amine analytes are performed using normal-phase systems containing mobile phases of heptane with ethanol (or 2-propanol) and an amine additive. Since salt-forms of amine analytes are usually insoluble in normal-phase eluents, free-base forms are synthesized for preparative chromatography. It would be highly desirable to directly chromatograph salt forms of amine analytes using mobile phases of carbon dioxide (CO(2)) and methanol (MeOH). Such separations would be readily suitable for preparative chromatography, since most amine salts are highly soluble in MeOH. In this article, advantages are shown for the use of supercritical fluid chromatography (SFC) instrumentation with tandem UV and polarimetric detection for confirming enantioseparation as well as for determining optimum preparative column injections. Examples are shown for racemic mixtures of propranolol HCl (I), thioridazine HCl (II), tramadol HCl (III), and flurbiprofen (IV), all of which resolved on Chiralpak AD-H chiral stationary phase using mobile-phase systems of CO(2) and MeOH without the use of basic or acidic additives.     

Analysis and purification of alcohol-sensitive chiral compounds using 2,2,2-trifluoroethanol as a modifier in supercritical fluid chromatography

2,2,2-Trifluoroethanol (TFE) is evaluated as an alternative modifier for the analysis and purification of alcohol-sensitive chiral compounds using supercritical fluid chromatography (SFC). Four chiral compounds, selected for their sensitivity to alcohols, in addition to a variety of standard chiral compounds were analyzed by SFC using TFE with polysaccharide and Pirkle-type chiral stationary phases (CSPs) to produce selectivities (alpha) and resolutions (Rs) as high as 1.4 and 7.2. A preparative isolation of 2-phenylglutaric anhydride was achieved using TFE as the mobile phase modifier to produce clean enantiomers.     

Effects of Hexane in Supercritical Fluid Chromatography for the Separation of Enantiomers

Supercritical fluid chromatography (SFC), operated in conventional mode, is normally recognized as normal phase chromatography, and uses a solvent combination of supercritical CO₂ and alcohols to separate compounds. Hexane, a commonly used solvent in normal phase liquid chromatography (NP‐LC), is rarely used in SFC and, in some cases, is added to the organic modifiers to increase liquid content in order to achieve better efficiency in preparative SFC for poorly retained compounds. Although hexane is believed to have similar solvent strength to that of supercritical CO₂, its effects on the enantioseparation in SFC is largely unknown. To understand the chromatographic effects of an apolar solvent, such as hexane in SFC, we compared the chromatographic behaviors of 35 chiral compounds using a parallel SFC method under traditional SFC mode of only “pure” alcohol‐CO₂ to that of hexane‐assisted SFC (HA‐SFC), which uses mixtures of alcohol and hexane (as cosolvents) and CO₂. We observed that, in some cases, hexane behaves just like supercritical CO₂, where replacement of a portion of CO₂ with hexane does not significantly change retention times or resolution of the peaks. In many cases, however, addition of hexane in mobile phases does affect chromatographic behavior of one or both enantiomers. Such effects might provide opportunities for separation of some enantiomers. Chirality 28:192–198, 2016. © 2016 Wiley Periodicals, Inc.     

Polar organic phase liquid chromatography with packed capillary columns using a vancomycin chiral stationary phase

Vancomycin immobilized on silica served as the chiral stationary phase (CSP) in this investigation with polar organic solvents as the mobile phase in liquid chromatography (LC). It was shown that trace amounts of water were beneficial for improving peak shape and efficiency. To regulate the retention and selectivity an acid and/or base were added to the mobile phase where an excess of acid was shown to be preferential for enantioseparation. An unusual increase in selectivity with increasing temperature was shown for the acidic drug, thalidomide. Additionally, nonlinear van't Hoff plots were obtained for metoprolol enantiomers that showed increased retention with increasing temperature. Metoprolol also showed unusual behavior in the polar organic phase when water was added to resemble reversed-phase chromatography, with minimum retention observed at high water or high methanol concentrations. In both instances a high degree of electrostatic interaction between metoprolol and vancomycin was concluded. Metoprolol and ten of its analogs were examined on this CSP to evaluate the enantiorecognition process. A comparison in enantioselectivity for a number of acidic and basic drugs using this CSP was also carried out using the polar organic phase, reversed phase, and normal phase LC which were all compared to the results obtained in supercritical fluid chromatography (SFC). Polar organic phase LC offered a better separation of basic molecules while reversed phase LC was preferred for the resolution of acids. SFC showed the broadest enantioselectivity overall and normal phase LC indicated similar properties, as expected, to SFC but with lower column efficiency. Copyright 2000 Wiley-Liss, Inc.     

Porous graphitic carbon shows promise for the rapid chromatographic analysis of polar drug metabolites

A rapid and simple HPLC method has been developed and used to separate the polar metabolic conjugates of AZT, chloramphenicol, and β-estradiol based upon the use of porous graphitic carbon. The HPLC system is sufficiently selective to resolve the polar drug conjugates from their parent compounds and from endogenous material present in urine. The compounds are separated, without the need for sample pretreatment or gradient elution, on a porous graphitic carbon (Hypercarb) column using aqueous trifluoroacetic acid modified with tetrahydrofuran as the mobile phase. Porous graphitic carbon exhibits a novel mechanism of retention towards these very polar substances, which are unretained under reversed-phase conditions on alkyl-bonded silica phases.     

Evaluation of the macrocyclic antibiotic LY333328 as a chiral selector when used as a mobile phase additive in narrow bore HPLC

The macrocyclic antibiotic LY333328 has been evaluated as a chiral selector for the enantioseparation of nine dansylated amino acids. This macrocyclic glycopeptide was used as a chiral mobile phase additive (CMPA) in conjunction with narrow bore high‐performance liquid chromatography (HPLC). The key mobile phase parameters of LY333328 concentration and buffer pH were varied, along with variations in stationary phases consisting of C8, phenyl, cyano, and silica. After observing and plotting changes in retention and resolution based on corresponding variation in these parameters, a better understanding of the behavior of this chiral selector was obtained. The pK_a values of the dansyl amino acid analytes and LY333328 were measured and used to gain a better understanding of the microenvironment in which these enantioseparations occur. Optimized conditions resulted in the baseline separation of eight of nine dansyl amino acids. Chirality 11:75–81, 1999. © 1999 Wiley‐Liss, Inc.     

Direct coupled column separation and determination of the diastereomeric glucuronides of almokalant, a new class III antiarrhythmic drug, in human urine.

A reversed-phase coupled column separation (CCS) system for the analysis of two diastereomeric glucuronides of almokalant, a new class III antiarrhythmic drug, in human urine is described. After direct injection of urine samples (50 microliters) the glucuronides were isolated by complex formation on a terbium(III) loaded strong cation exchanger at alkaline pH. The solutes were eluted from the precolumn by an acidic mobile phase, enriched and separated on Hypercarb (porous graphitic carbon) as analytical column with 0.1 M acetic acid pH 2.8 and 30% acetonitrile as mobile phase. The calibration graph was linear (r2 = 0.9999) and the detection limits were in the low picomole (UV) or femtomole (fluorescence) range. Optimization of the analytical column revealed that elution order and selectivity for the glucuronides were dependent on the buffer agent and temperature used. By appropriate choice of mobile phase conditions all four diastereomers could be separated.     

Use of a naphthylethylcarbamoylated-β-cyclodextrin chiral stationary phase for the separation of drug enantiomers and related compounds by sub- and supercritical fluid chromatography

Enantiomeric separation of a variety of drugs and related compounds was achieved on an (S)-naphthylethylcarbamoylated-β-cyclodextrin (S-NEC-CD) chiral stationary phase (CSP) using sub- and supercritical fluid chromatography (SFC). Compounds previously resolved on native or derivatized cyclodextrin CSPs in liquid chromatography (LC) using reversed phase or polar organic mobile phase modes could be resolved in SFC using a simple carbon dioxide/methanol eluent. Resolution of cromakalim, which is not possible on the S-NEC-CD column in LC, was readily accomplished in SFC. The importance of modifier, temperature, and pressure was assessed in relation to retention, selectivity, and resolution. The nature of the modifier and the modifier concentration were found to be crucial parameters. © 1996 Wiley-Liss, Inc. Contribution of the National Institute of Standards and Technology. Not subject to copyright.     

Packed-column supercritical fluid chromatography for the analysis of isosorbide-5-mononitrate and related compounds in bulk substance and tablets

We describe a packed-column supercritical fluid chromatographic method that can be used for the analysis of isosorbide-5-mononitrate (5-ISMN) bulk substance and the 5-ISMN content of Imdur tablets. The method is based on methanol-modified carbon dioxide as the mobile phase and porous graphitized carbon (PGC, Hypercarb) as column support at 40 degrees C and 100 bar back pressure. The method makes it possible to simultaneously determine 5-ISMN and related compounds. In order to elute NO(3)(-) with acceptable retention time a quarternary ammonium hydrogen sulfate salt is added to the methanol modifier. An almost linear increase of the retention time with increasing carbon content of the counter ion was found. Tetramethyl ammonium hydrogen sulfate 5 mM in methanol was used in the final method as polar modifier for the simultaneous determination of possible degradation products within 12 min. The present method can separate and detect related compounds such as isosorbide-2, 5-dinitrate, isomannidemononitrate and isosorbide-2-mononitrate at the 0.1% (w/w) level as required by regulatory guidelines. Nitrate can be detected down to about 0.02% (w/w). Repeated analyses of ground tablet powder gave an assay precision for isosorbide-5-mononitrate of 1.4% (R.S.D., eight samples and two injections of each). For related substances at an area percent of 0. 1 the precision was less than 10%.     

Tuning of mobile and stationary phase polarity for the separation of polar compounds by SFC

The separation of polar compounds by supercritical-fluid chromatography (SFC) is reviewed. New developments in mobile and stationary phase tuning are reviewed for packed and packed capillary SFC. In terms of mobile phase polarity adjustment, new pure and multiple component fluids are presented. The approach of tuning the polarity of the stationary phase as a way of increasing the range of polar compounds analyzed by SFC using pure CO(2) is discussed using either silica-based or new materials as stationary phase. Chiral, liquid crystal and polymer-based stationary phases coated on particles are widely covered in this review as an interesting approach to separate polar compounds avoiding the major drawbacks associated to the use of modifiers in SFC.     

Simulated moving bed chromatography with supercritical fluids for the resolution of bi-naphthol enantiomers and phytol isomers

The combination of the simulated moving bed (SMB) technique with supercritical fluid chromatography (SFC) leads to a process with unique features. Besides the known advantages of the SMB process, the use of supercritical carbon dioxide as the mobile phase offers the advantages of reduction in organic solvents and an easy eluent/solute separation. Because of the low viscosity and high diffusion coefficients of supercritical fluids, a high efficiency is possible. The steps of process development for SMB SFC are presented using the separations of the bi-naphthol enantiomers and phytol isomers as examples. The development of a packed column SFC method at an analytical scale is shown for the separation of the bi-naphthol enantiomers on a chiral stationary phase and CO(2) with a modifier as the mobile phase. The influence of the modifier, modifier content, and column configuration on productivity of the SMB SFC process was investigated by simulation. The first set of experiments was performed in the SMB separation of phytol isomers at low concentration to test the feasibility of the SMB SFC high purity separation of the binary mixtures. In the second set of experiments, the productivity of the process was increased by increasing the feed concentration up to 54 grams feed per liter stationary phase (SP) and hour (g(feed)/l(SP) h).     

Direct high-performance liquid chromatographic separation of the enantiomers of an aromatic amine and four aminoalcohols using polysaccharide chiral stationary phases and acidic additive

The HPLC enantiomeric separation of N-benzyl-alpha-methyl-benzylamine, phenylalaninol, tryptophanol, 2 (diphenylhydroxymethyl)pyrrolidine, and isoproterenol was accomplished in the normal-phase mode using two polysaccharide-derived chiral stationary phases (CSPs) and various n-hexane/2-propanol mobile phases with acidic (TFA) or basic (DEA) additive. The compounds were separated without any derivatization and separation factor range between 2.09 and 1.09 with resolution factor 3.4 and 0.4, respectively. The best separation of the enantiomers of the amine was achieved on amylose tris (3, 5-dimethylphenylcarbamate) CSP with TFA additive in the mobile phase; in acidic conditions, instead, the best enantioseparation of the aminoalcohols was achieved on cellulose tris (3, 5-dimethylphenilcarbamate). A long equilibration time of the CSP when switching from an undoped mobile phase to a doped one is required to obtain reproducible results.     

Manipulation of chiral resolution for isoxazoline-based IIb/IIIa receptor antagonists using various mobile phase additives in subcritical fluid chromatography

Subcritical fluid chromatography (SubFC) using a carbon dioxide-methanol mobile phase is used for the chiral resolution of IIb/IIIa receptor antagonist enantiomers. The chiral resolution of three analogs, each containing two chiral centers, is optimized using various mobile phase additives. The effects that acidic, basic, and neutral additives have on retention, efficiency, and resolution are examined. The additive that gives the best resolution was found to be dependent upon the functionality and charge of the chiral analyte. For charged analytes, additives that act as competing ions of the same charge as the chiral analyte dramatically improve efficiency and resolution. Resolution of neutral chiral analyte enantiomers is also greatly affected by the choice of mobile phase additive. Chirality 10:338–342, 1998. © 1998 Wiley-Liss, Inc.     

Direct enantiomeric separation of beta-blockers on ChyRoSine-A by supercritical fluid chromatography: supercritical carbon dioxide as transient in situ derivatizing agent.

The direct enantiomeric separation of a series of beta-blockers has been carried out on two chiral stationary phases (CSPs) derived from 3,5-dinitrobenzoyl tyrosine: the commercially available ChyRoSine-A and a recent improved version of this CSP. Using supercritical fluid chromatography (SFC), facile separations are achieved (1.1 less than Rs less than 7) within short analysis times. The parameters affecting the enantioselectivity (temperature, pressure, mobile phase nature, solute structure) have been investigated. The optimal mobile phase consists in a mixture of carbon dioxide-methanol-propylamine at 25 degrees C. The solute structure has a great influence on the enantioselectivity. For instance, both amine and hydroxyl protons are necessary for chiral discrimination to occur. Furthermore, the steroselectivity value is directly connected to the amine substituent steric bulkiness. Surprisingly, these solutes are poorly resolved using normal phase liquid chromatography (NPLC). Accordingly, the specific influence of carbon dioxide on the enantiomeric separation of 1,2-amino-alcohols have been investigated using various techniques such as nuclear magnetic resonance (NMR) or molecular modelisation. It has been shown that carbon dioxide acts as a complexing agent toward the amino-alcohol by setting up of a bridge with the hydroxyl and the amine protons of the solute. In that way, the resulting complex possesses lower acido-basic properties and a higher conformational rigidity, responsible for chiral discrimination.     

Enantiomeric Separation of Original Heterocyclic Organophosphorus Compounds in Supercritical Fluid Chromatography

New and original heterocyclic α-enamido phosphine chiral solutes were prepared: four structurally similar racemates with the chirality center placed on the phosphorus atom, and four other related pairs of enantiomers with chirality borne by the carbon atoms of the phospholane ring. The structural variations were placed on an aliphatic heterocycle (six- or seven-member rings) and on the carbamate function (methyl or t-butyl). Their separation was achieved on a commercial cellulose tris-(3,5-dimethylphenylcarbamate) stationary phase (Lux Cellulose-1, Phenomenex) in supercritical fluid chromatography (SFC). The effects of molecular structure on SFC retention and enantioresolution were studied. Among these eight pairs of enantiomers, some reversal of elution order between similar compounds was observed. The effect of changing the organic solvent (methanol and ethanol) and its proportion (between 5 and 40%) in the mobile phase was investigated. Retention data were collected over the temperature range 0–50 °C, and the results interpreted from thermodynamic aspects. Chirality, 2013. © 2013 Wiley Periodicals, Inc.     

Enantiomer separation by complexation SFC on immobilized Chirasil-nickel and Chirasil-zinc

The use of complexation SFC for enantiomer separation of Lewis base selectands on chiral nickel(II)- and zinc(II)-bis[(3-heptafluorobutanoyl)-10-methylene-(1R)-camphora te] chemically bonded to poly(dimethylsiloxane) (Chirasil-nickel and Chirasil-zinc) and employed as Lewis acid selectors is described. The method is especially suited for less volatile and configurationally labile racemates. The variation of the experimental parameters temperature T, pressure p and density rho of the mobile phase carbon dioxide on the retention factor k, relative retention r and chiral separation factor alpha is studied, providing insights into the mechanisms of chiral recognition under supercritical conditions. For mecoprop methyl ester (methyl 2-(4-chloro-2-methylphenoxy)propanoate) an unusual increase of alpha at increased temperature is observed on Chirasil-nickel. Supercritical carbon dioxide does not inadvertently affect the complexation equilibria between Lewis donor selectands and the Lewis acid metal selectors during complexation SFC.     

Evaluation of a ristocetin bonded stationary phase for subcritical fluid chromatography of enantiomers

A stationary phase derived from ristocetin was evaluated for chiral separation in subcritical fluid chromatography. Separation of various enantiomers having different structures and pK(a) values were investigated using carbon dioxide and polar modifiers. The influence of modifiers, additives, temperature, and mobile phase flow rate on separations is presented. It is concluded that this stationary phase can be used for SFC despite its structural similarity with protein-derived stationary phases that can only be used in HPLC. The separation mechanisms could not be elucidated or predicted using these initial experiments. The separations of warfarin and, especially, efavirenz demonstrate the potential of this type of stationary phase for rapid SFC chiral separations.