Achiral Stationary Phases Research Articles

Chromatographic and Mass-Spectrometric Studies of Cyclic 2-(N,N-Dimethylaminomethyl)ferroceneboronates and Related Esters

Cyclic 2-(N, N-dimethylaminomethyl)ferroceneboronate (DMAM-FcB) esters of a variety of 1, 2-diols have been examined by gas and liquid chromatography as well as by mass spectrometry. Some comparisons have been made with analogous ferroceneboronate (FcB) esters. Under the conditions used for reversed-phase high-performance liquid chromatography, more of the FcB than of the DMAM-FcB esters were stable. Many of the derivatives were suitable for gas chromatography; the retention index increments for the dimethylaminomethyl substituent were on the order of 200 units on a methylsilicone phase. A satisfactory separation of diastereomeric boronates derived from chiral diols could be performed in the same achiral stationary phase. In most instances, the mass spectra of DMAM-FcB esters showed clear molecular ions along with prominent ions of m/z (M-44), resulting from a loss of the (CH3)2N radical. Evidence for the formation of mono- and bis-boronates of ecdysterone was obtained by fast atom bombardment mass spectrometry.

Enantiomeric derivatization for biomedical chromatography.

Derivatization reactions aimed at creating the basis for the chromatographic resolution of biologically and pharmaceutically important enantiomers are reviewed, with emphasis on the literature published in the last 10 years. Three main aspects of chiral derivatization are discussed. (a) Enantiomers containing suitable functional groups (amino, carboxyl, hydroxyl, epoxy, etc.) are transformed into covalently bonded diastereomeric derivatives using homochiral derivatizing agents. The diastereomers formed (esters, amides, urethanes, urea and thiourea, etc., derivatives) can be separated on achiral stationary phases. The derivatization reactions often afford further advantages, such as the improvement of chromatographic properties and the detectability of the solutes using UV and fluorimetric detectors. (b) Covalent but achiral derivatization is often necessary even with the use of chiral stationary phases enabling in principle direct enantioseparations (Pirkle-type columns, cyclodextrin-bonded phases, glycoprotein column and functionalized cellulose columns). The main goals of these derivatization reactions (which are analogous to those discussed above), are to introduce functional groups into the molecule of the enantiomers that improve the possibilities for chiral interactions or block functional groups to avoid non-specific interactions. (c) In the broader sense, the dynamic formation of diastereomers using chiral mobile phase additives (cyclodextrins, various reagents to form diastereomeric ion pairs, adducts, mixed metal complexes) can also be considered to be chiral derivatization reactions and is therefore briefly discussed also.

HPLC Separation of the ZZ, ZE, EZ, and EE Geometric Isomers and EE Isomer Enantiomers of a Substituted Pentadienyl Carboxamide Using Achiral/Chiral Column Switching

Abstract The four geometric isomers of a substituted pentadienyl carboxamide were separated on an achiral aminopropyl column coupled with a silica precolumn. The R and S enantiomers of the biologically active EE isomer (RO 24–0238 and RO 24–2099) were resolved (Rs 1.65) on a cellulose-based chiral stationary phase (Chiracel OF). The coupled silica and aminopropyl columns were connected to the Chiralcel OF column through a six port switching valve that enabled transfer to the EE isomer to the chiral phase for enantiomer resolution. By examining the selectivity for separation of the geometric isomers of various achiral stationary phases using hexane-isopropanol mobile phases, a method was developed which linked the achiral separation of the geometric isomers with the chiral separation of the EE enantiomers.

Enantioselective Ion-Pair Chromatography of Phenolic 2-Dipropylaminotetralin Derivatives on Achiral Stationary Phases: an Experimental and Theoretical Study of Chiral Discrimination.

Enantioselective Ion-Pair Chromatography of Phenolic 2-Dipropylaminotetralin Derivatives on Achiral Stationary Phases: an Experimental and Theoretical Study of Chiral Discrimination.

Determination of leucovorin and 5-fluorouracil in plasma by high-performance liquid chromatography.

A method was developed for the determination of (6R)- and (6S)-leucovorin and 5-fluorouracil in plasma. As leucovorin diastereoisomers cannot be separated on a classical reversed-phase column, it was necessary to use a chiral stationary phase. The method presented is based on the same principle as the method described by Wainer and Stiffin [J. Chromatogr., 424 (1988) 158], i.e., coupling of a bovine serum albumin phase to an achiral stationary phase. Before the chromatography, the drug was isolated from the plasma matrix by solid-phase extraction. For 5-fluorouracil, chromatography was performed on a classical RP-18 column after extraction from the plasma by liquid-liquid extraction. Both methods were validated and applied to the analysis of patients' samples.

Benzoylcellulose derivatives as chiral stationary phases for open tubular column chromatography

AbstractThe application of cellulose‐based stationary phases for chiral separations has been extended to open tubular column chromatography. Efficient columns were obtained by coating the capillaries with mixtures of chiral cellulose materials and conventional achiral stationary phases for gas chromatography. In this study, various siloxane and polyethylene glycol polymers were used as achiral components and mixed with different substituted benzoylcellulose derivatives as chiral components. Systematic investigations were carried out to determine the optimal ratio for the components of the stationary phase. Depending on the chromatographic mode—gas chromatography (GC) or supercritical fluid chromatography (SFC)—the stationary phases were found to behave differently. The applicability of the technique was demonstrated by the resolution of various racemic compounds. © 1993 Wiley‐Liss, Inc.

Gas chromatographic behaviour of isomeric tricyclo[10.4.0.04,9]hexadecanes

Using achiral stationary phases of different polarity in capillary gas chromatography it is demonstrated that the hydration of 1,2–5,6-dibenzocycloocta-1,5-diene yields five isomers of the tricyclo[10.4.0.04,9]hexadecanes. With highly polar stationary phases all isomers of the tricyclic hexadecanes and the semihydrated products in the mixture were also separated from each other. The chiral isomers were separated on cyclodextrin stationary phase. Moreover, with columns of achiral and chiral phases coupled in series, in a double oven gas chromatograph, it was possible to estimate the configuration of the isomers. The separation of diastereomers on chiral stationary phases is a useful means for structural assignment of isomeric compounds.

Computer simulation of three scenarios for the separation of non-racemic mixtures by chromatography on achiral stationary phases

With the aid of a general chromatography simulation program based on the theoretical plate model, three scenarios rationalizing the phenomenon of the separation of non-racemic mixtures into racemate and excess enantiomers by chromatography on achiral stationary phases were simulated. The separation, repeatedly observed in high-performance liquid chromatography and gas chromatography, can be explained by the formation of homo- and heterochiral self-associates ( i.e. RR, SS and RS). The kinetic calculations were performed using a Runge—Kutta routine. This program offers for the first time a general means of simulating dynamic chromatographic elution profiles involving any type of reaction occurring during chromatographic separation. Thus computer simulation of experimental elution profiles is no longer limited to first-order reactions such as enantiomerizations or isomerizations.

Resolution of Enantiomers with Achiral Phase Chromatography

Abstract Resolution of enantiomers is of widely accepted importance. Since there have been sporadic reports on liquid chromatographic resolution of enantiomers using achiral stationary phase in conjunction with a mobile phase having no external chiral compound the present paper focuses on such achiral phase resolutions of enantiomers along with their possible explanations. At the same time it introduces to the present day practice of resolution of enantiomers by liquid chromatography which makes use of a chiral selector either bound to the stationary phase or added to the mobile phase. The mechanisms for chiral phase chromatographic methods are also discussed.

Chiral separation on achiral stationary phases with different functionalities using β-cyclodextrin in the mobile phase and application to bioanalysis and coupled columns

β-cyclodextrin was used in the mobile phase as chiral selector for separating the enantiomers of terbutaline, chlorthalidone and oxazepam. The effect on chiral resolution using e.g. hydrophobic, polar or cation exchanging stationary phases was investigated. Both the chiral separation factor and retention level were affected by the concentration of methanol and β-cyclodextrin. The stationary phase had no effect on the chiral separation only on the level of retention. By tuning the concentration of β-cyclodextrin and methanol in the mobile phase chiral separation could be obtained on most stationary phases. By changing the stationary phase while adjusting the mobile phase composition to maintain the chiral selectivity, improvements of the selectivity towards e.g. endogenous compounds can be obtained when separating enantiomers in complex matrixes as biological fluids. Further improvement on selectivity can be obtained if coupled columns are used. This is examplified for separation of chlorthalidone and terbutaline enantiomers in biological fluids by coupling an achiral column to another achiral column and using a mobile phase containing β-cyclodextrin on the last column.

Liquid and high-pressure carbon dioxide chromatography of β-blockers : Resolution of the enantiomers of nadolol

The enantiomers of six chiral aryloxypropanolamine β-blockers were separated by high-performance liquid chromatography on a column of modified cellulose immobilized onto silica (Chiralcel OD), using mixtures of heptane or hexane with a polar modifier. The quality of the separations was improved and retention times were reduced by eluting under subcritical conditions with carbon dioxide containing 20% of methanol. Nadolol, which has two chiral centres, gave three peaks instead of four on Chiralcel OD. A separation of the four isomers was obtained on α 1-acid glycoprotein column eluted with an aqueous buffer containing octanoate ions. A direct separation of the two diasteromers could not be obtained with achiral stationary phases, but the derivative formed by the base-catalysed reaction of nadolol with isopropyl isocyanate was well resolved on a column of amino-bonded silica eluted with carbon dioxide—methanol. This paper demonstrates the advantages of high-pressurecarbon dioxide chromatography for the analysis of stereoisomers.

Gas chromatographic separation of diastereomeric esters of α-alkyl-α-amino acids on dimethylpolysiloxane

The separation of 28 ( R,S)-α-alkyl-α-amino acids (AAAs) with the structure H 2NC(R 1)(R 2)COOH (R 1,R 2 = alkyl, aralkyl, which may be further substituted; R 1,R 2 ≠ H) was systematically investigated by capillary gas chromatography on the stationary phase dimethylpolysiloxane (CP-Sil 5); the AAAs were used as their N(O)-pentafluoropropionyl (PFP)-AAA ( S)(+)-2-alkyl esters (alkyl = 2-butyl to 2-octyl and 3-methyl-2-butyl). In selected cases, ( R,S)-3,3-dimethyl-2-butyl esters, ( R,S)-3-octyl esters, ( R,S)-2-dodecyl esters and (−)-menthyl esters were also employed. Baseline resolution was achieved for these AAAs by derivatization with suitable alcohols. By using AAAs of known absolute configuration, it was possible to establish the order of emergence as ( S)-AAAs before ( R)-AAAs for PFP-( R,S)-AAA ( S)(+)-2-alkyl esters. This is the opposite elution order to that of α-amino acids having a C α-hydrogen atom. In spite of their opposite elution orders on achiral stationary phases, conformational analysis of PFP ( S)(+)-2-alkyl esters of ( S)-α-amino acids and ( S)-AAAs shows that they have the same bulkiness chirality.

Diastereoisomeric Derivatization and Liquid Chromatographic Analysis of N-(Phenylsulfonyl)-2-phenylglycine Aldose Reductase Inhibitors

A series of (S)- and (R)-N-(phenylsulfonyl)-2-phenylglycines are synthesized as potential inhibitors of the enzyme aldose reductase. In vitro analysis of these compounds reveals that the S-enantiomers are more potent than the corresponding R-enantiomers and that the difference in potencies between enantiomeric pairs is dependent on the nature of the ring substituent. To ensure that the enantioselectivity observed does not reflect varying degrees of racemization during the synthesis of the N-(phenylsulfonyl)-2-phenylglycines, the enantiomeric purity of these products is determined by HPLC after chiral derivatization. Each 2-phenylglycine inhibitor is derivatized with R-alpha-methylbenzylamine, and the resulting diastereomers are analyzed using reversed and normal achiral stationary phases. Reversed-phase methods with C18 or phenyl stationary phases and solvent mixtures of acetonitrile or methanol in water do not provide satisfactory resolution of the diastereomers. However, normal-phase analyses with a silica stationary phase and mixtures of methanol, ethanol, or acetonitrile in chloroform provide good separations with relatively short analysis times. The normal-phase analyses demonstrate that a single diastereomeric amide forms from each N-(phenylsulfonyl)-2-phenylglycine product, establishing that these compounds do not racemize during synthesis.

Chromatographische Trennung und quantitative Bestimmung aller acht Stereoisomeren von α‐Tocopherol

Separation and Quantitation of All Eight Stereoisomers of α‐Tocopherol by ChromatographyThe results of the analysis of several derivatives of (all‐rac)‐α‐tocopherol by HPLC on a chiral stationary phase and by capillary GC on a achiral stationary phase are reported. Consecutive application of both methods to the ethyl‐ether derivative allows the separation and quantification of all eight possible stereoisomers of (all‐rac)‐α‐tocopherol.

Chromatographic Separation of the Excess Enantiomer under Achiral Conditions

The excess enantiomer can be separated chromatographically without difficulty from mixtures of enantiomers in the ratio a: b (a ≠ b), as are usually formed in enantioselective syntheses. This is possible on achiral stationary phases and with achiral mobile phases, but only in utilizable amounts if both homochiral as well as heterochiral associates are formed in solution. These associates are diastereomeric. Hence, the stationary phase is of decisive importance. On aminopropyl silica gel the excess entiomer is eluted first, then the racemate.

π-acceptor amide group for liquid chromatographic chiral separations with special emphasis on the 3,5-dinitrobenzoyl amide

A series of achiral π-acceptor benzene derivatives and of achiral benzamide derivatives were chromatographed on a chiral and an achiral stationary phase to study the specific interactions of π-acceptor samples with a chiral π-donor stationary phase. The specific interactions with the chiral moiety of the stationary phase are expressed by the relative selectivity k′* [ k′* = k′ (chiral phase)/ k′ (achiral phase)]. To study the chiral recognition, a series of chiral phenylethylamine derivatives were chromatographed on the same two phases to determine the influence of the π-acceptor amide group on the chiral separation. The separation factor α does not correlate with the capacity factor k 2′ but it correlates with the relative selectivity k 2′*. If the aromatic moiety of the π-acceptor is flat, there is a correlation of the AMPAC-calculated LUMO (lowest unoccupied molecular orbital) with the relative selectivity k′*. The 3,5-dinitrobenzoyl group ideally combines flatness with a low LUMO.

Separation of enantiomers in thin-layer chromatography

Up to now in thin-layer chromatography a series of retention mechanisms for the determination of enantiomeric compositions have been applied. Subject of this paper is a corresponding survey with a focal point on ligand exchange chromatography. In connection with this retention mechanism most successfully used in TLC for the separation of optical antipodes, some additional findings of the authors are included. Retention mechanisms in question are: - Formation of inclusion complexes within the cavities of cyclodextrins. Substance classes separated by this system are e.g. dansyl amino acids or metallocenes. - Interaction with so-called “brush type” phases. In this case, chiral groups with π-acceptor of π-donor properties are chemically or physically bonded to an aminomodified silica gel matrix. Chiral compounds, like aromatic alcohols, were separated on these stationary phases. - Differences in retention of diastereomers formed previously or during the chromatographic process on an achiral stationary phase. Some examples for the manifold applicability of this principle are the separation of chiral amines from biological matrices, hydroxy-fatty acids, and amino acids. - Selective interactions with the helical structures of cellulose or cellulose triacetate. Referring to this technique, separations of several amino acids and of the analgesic agent Oxidanac in the R-and S-species are described. - Enantioselective ligand exchange, based on reversed phase silica gels impregnated with copper salts and optically active amino acid derivatives. In thin-layer chromatography this is the most important principle used mainly for the separation of racemic mixtures of free amino acids and amino acid derivatives. The recently developed HPTLC pre-coated plate CHIR with concentrating zone is also based on this retention mechanism. Aside from the applications mentioned above, dipeptides, hydroxy-carboxylic acids, α-halogenated carboxylic acids, amino lactones, and thiazolidine derivatives have been separated on this plate up to now. Furthermore, this new pre-coated layer is particularly suitable for an optical in situ evaluation and quantitative determinations of optical antipodes.

Separation of the enantiomers of substituted putrescine and cadaverine analogues by gas chromatography on chiral and achiral stationary phases

Capillary gas chromatography (GC) on chiral stationary phases, i.e., Chirasil-Val [ l-valine- tert.-( R)-α-butylamide] and XE-60- S-valine-( R-α-phenylethylamide, has been applied to the resolution of various substituted analogues of putrescine as their N,N′-perfluoroacyl derivatives. The influence of the nature of the substituent on the retention behaviour and on the resolution of the enantiomers was studied. The results are discussed in terms of volatility analogues with two chiral centres were also clearly resolved into their corresponding stereoisomers. When the chain length between the two amino groups was increased, no clear resolution was obtained of the monosubstituted cadaverine analogues as their N,N′-perfluoroacyl derivatives. However, resolution was obtained after derivatization of the cadaverine analogues with ( — )-α-methoxy-α-trifluoromethylphenylacetyl chloride, followed by GC analysis on an achiral phase.

Liquid chromatographic determination of the enantiomeric composition of amphetamine prepared from norephedrine and norpseudoephedrine.

The stereochemical course of the synthesis of amphetamine from norephedrine and norpseudoephedrine is investigated using liquid chromatography. The results show that the chiral carbon common to both compounds remains unaffected during the reaction sequence. The presence of individual amphetamine enantiomers in the reaction products is determined by reversed-phase liquid chromatographic separation on an achiral stationary phase (C18) following precolumn derivatization with 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (GITC). The GITC derivatization procedure allows for the liquid chromatographic separation of the individual enantiomers of amphetamine, norephedrine, norpseudoephedrine, and the intermediate 1-chloro-1-phenyl-2-aminopropanes.

Separation of dipeptide diastereomers by high-resolution gas chromatography

Separation of trimethylsilylated diastereomers of dipeptides was achieved by high-resolution gas chromatography on a fused silica capillary column coated with an achiral (conventional) stationary phase. l, i and d, d forms (enantiomers) were separated from their diastereomers, l, d and d, l with excellent resolution. Enantionmers were not separable from each other ( l, l from d, d and l, d from d, l) on this column. The nominal structures of the separated stereoisomers were confirmd by gas chromatography—mass spectrometry.