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

Summary -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 -cyclodextrin. The stationary phase had no effect on the chiral separation only on the level of retention. By tuning the concentration of -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 -cyclodextrin on the last column.     

Liquid–Gas Adsorption Chromatography

The history of the discovery, physical and chemical properties, and analytical potentialities of chromatographic methods using the stationary gas phase, such as liquid–gas and liquid–gas adsorption chromatography, are considered. Taking into account the retention of the stationary gas phase in the pores of a hydrophobic support, one can optimize the conditions for the separation and extraction of volatile substances from aqueous solutions on hydrophobic adsorbents in solid-phase extraction and reversed-phase liquid chromatography. Scientific progress is the progress of a method. M.S. Tswett     

Enantiomer separation of side-chain fluorinated alkylbenzenes by capillary gas chromatography on cyclodextrin phases

Summary The separation of racemic side-chain fluorinated alkylbenzenes and bromofluorinated analogues by capillary gas chromatography using permethylated , and -cyclodextrins dissolved in polysiloxanes of different polarity as stationary phases is described. The influence of the achiral polysiloxane matrices on the separation of enantiomers is discussed in the light of the results obtained with the different phases. For a part of the tested compounds thermodynamic data are determined which describe the interaction of enantiomers with the stationary phase. The mechanism of separation is discussed on this basis and by comparison with data for structurally similar compounds.     

Comparative study of some commercial chemically bonded phases in classical reversed-phase chromatography and in ion-pair reversed-phase liquid chromatography

Summary The behaviour of various octadecyl commercial bonded phases are compared in classical reversed-phase chromatography and in ion-pair reversed-phase chromatography. Great differences are exhibited by the packings studied according to the polarity of the solutes. Whereas hydrocarbonaceous bonded phases show very similar selectivity versus apolar or weakly polar solutes, great differences are observed when analyzing more polar solutes even when ion-pair reversed-phase chromatography is performed.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Reversed phase liquid chromatographic retention behavior of polystyrene homopolymers

Summary Isocratic and gradient elution high performance liquid chromatographic measurements of the retention behavior of polystyrene homopolymers with molecular weights ranging from 2 kD to 390 kD were performed using mixed methylene chloride-methanol mobile phases of varying composition and a C-18 chemically bonded stationary phase supported on either 100 Å or 300 Å mean pore diameter silica. Isocratic measurements of the capacity factor, k, for different molecular weight homopolymers as a function of the volume fraction of methylene chloride, , permit determination of the critical composition, _c, which renders k=1 and the local slope, S=–lnk/_c of the lnk- isotherm at =_c, and also the dependence of _c and S on the degree of polymerization, M. Linear gradient elution measurements of _c and S were also performed and compared to the corresponding isocratic measurements. The general retention behavior and the dependence of _c and S on M compare favorably to the predictions of the theory of homopolymer retention and fractionation developed by Boehm, Martire, Armstrong, and Bui (BMAB). Comparison is also made between the present work and the experimental observations of other workers on related chromatographic systems involving hompolymer retention.     

Mobile phase effects in reversed-phase chromatography VI. Thermodynamic models for retention and its dependence on mobile phase composition and temperature

Summary The physico-chemical framework is examined by comparing the predictions of three models for the combined effects of the composition of the hydroorganic mobile phase and the column temperature on the retention ofn-alkylbenzenes on hydrocarbonaceous bonded stationary phases. The well-mixed model leads to expressions for the dependence of retention on three factors which are equivalent to those derived previously from linear extrathermodynamic relationships. The diachoric model stems from the assumption that the mobile phase is microscopically heterogeneous and the displacement model is identical to the retention model most widely used in chromatography with polar sorbents and less polar solvents. Over limited ranges of mobile phase composition and temperature, each model does describe retention behavior. However, only the wellmixed model describes retention well over the entire range of mobile phase composition and temperature studied here. The success of the well-mixed model, and its limits, give insight into the role of the organic solvent in determining the magnitude of chromatographic retention on non-polar stationary phases with hydro-organic eluents.Dedicated to Professor S. R. Lipsky on the occasion of his 60th birthday.     

Chiral chromatography of amino- and hydroxy-acids on surface modified porous graphite

Summary A new type of chiral stationary phase has been prepared by coating porous graphite with a near-monolayer of an adsorbed enantiomeric modifier which then acts as an adsorbed stationary phase. The most effective modifiers are L- or D-isomers of N-(2-naphthalene-sulphonyl)-phenylalanine (NS-Phe). Conveniently 80% of monolayer coverages, corresponding to 1.2 mol m^–2, are achieved by adsorption of NS-Phe from methanolic solution. Enantiomeric separations by complexation with cupric ion show base-line resolution of -amino and -hydroxy acids with separation factors up to 2. Identical separations are achieved with chiral phases made from the L- and D-isomers of NS-Phe except that the elution orders of enantiomers are inverted. Reduced plate heights are around 5 and the adsorbed chiral phases are extremely stable, retention ratios being unchanged after passage of 7000 column volumes of eluent. A mechanism of retention is proposed, which fits the experimental observations.     

Chromatographic investigations into the interaction between proteins and polymer matrices

Summary Chromatographic investigations with two enzymes and selected low-molecular-weight compounds of different pk_a on three different inert gels (matrices) commonly used for enzyme separation and immobilization showed that considerable deviations from the theoretical elution volumes occur at low ionic strength. These deviations are explained in terms of ionic and hydrophobic interaction between gel matrix and solute.     

Cyclodextrin- and sugar-derivatives as stationary phases for the gas chromatography of semivolatiles

Cyclodextrins as cyclic hexa-() to octa-() saccharides are widely and successfully used after esterification or etherification as stationary phases in gas chromatography for the separation of chiral compounds. Their basic potential as thermostable stationary phases as such or after derivatisation offers a unique spectrum of interactions ranging from hydrogen bonding to shape selectivity. Similar properties as thermostable stationary phases are shown by long-chain saccharide ethers (chain-length C8–C20), which are normally used as non-ionic detergents.     

Equilibrium between α- and β-isomers of dihydroartemisinine and its multiple-peaks in high-performance liquid chromatography

Summary The interconversion between the - and -isomers and the multiple-peaks of dihydroartemisinine (dihydroqinghaosu) were investigated in reversed-phase, highperformance liquid chromatography. A convenient method using the Gaussian distribution function instead of the Schmidt graphical method for the theoretical calculation of the diffuse probability curves versus the fraction of the dead time on column as form was introduced. Based on the theoretical probability curves and the experimental multiple-peak chomatograms, the effects of temperature and flow velocity on the accuracy and precision of analysis of dihydroartemisinine are indicated and the activation energy of the interconversion between - and -isomers of dihydroartemisinine is estimated.     

Comparison and characterization of reversed phases

Summary A pragmatic test procedure for comparison and evaluation of reversed phases is described. The differences in retention between RP8 and RP18 can be compensated for by adjustment of the eluent composition. A 10% increase in the water content doubles the k values, as does the exchange of an RP8 with an RP18 column. It is possible to differentiate between the two columns with the pair ethylbenzoate and toluene. Under stand-ard conditions (methanol-water, 55–45, v/v; 49–51, w/w) with RP8 ethylbenzoate is always eluted together or after toluene, whereas with RP18 it is always eluted in front of toluene.With the same eluent composition the suitability of stationary phases for the separation of basic solutes can be evaluated. With good phases—symmetrical peaks for basic solutes—aniline is always eluted before phenol and the peak asymmetry relationship of the aniline and phenol peak is less than 1.3. With such good stationary phases the retention of bases is independent of sample size if the linear capacity of 0.1 mg sample/g stationary phase is not exceeded. The test can also be used to study column stability towards hydrolysis.     

Insights into the retention mechanism of neutral organic compounds on polar chemically bonded stationary phases in reversed-phase liquid chromatography

The solvation parameter model is used to characterize the retention properties of a 3-aminopropylsiloxane-bonded (Alltima amino), three 3-cyanopropylsiloxane-bonded (Ultrasphere CN, Ultremex-CN and Zorbax SB-CN), a spacer bonded propanediol (LiChrospher DIOL) and a multifunctional macrocyclic glycopeptide (Chirobiotic T) silica-based stationary phases with mobile phases containing 10 and 20% (v/v) methanol-water. The low retention on the polar chemically bonded stationary phases compared with alkylsiloxane-bonded silica stationary phases arises from the higher cohesion of the polar chemically bonded phases and an unfavorable phase ratio. The solvated polar chemically bonded stationary phases are considerably more hydrogen-bond acidic and dipolar/polarizable than solvated alkylsiloxane-bonded silica stationary phases. Selectivity differences are not as great among the polar chemically bonded stationary phases as they are between the polar chemically bonded phases and alkylsiloxane-bonded silica stationary phases.     

Retention characteristics of octadecylsilyl silica,trimethylsilyl silica and phenyldimethylsilyl silica in reversed-phase liquid chromatography

Summary The hydrophobic retention characteristics of stationary ligands for reversed-phase high-performance liquid chromatography have been evaluated from the slope (r-value) of the plots relating the capacity factors (log k) of selected aliphatic and aromatic compounds with the reciprocal of methanol concentration (log (1/[MeOH])) in aqueous mobile phase. Octadecylsilyl (ODS), trimethylsilyl (TMS) and phenyldimethylsilyl (phenyl) groups were selected as the stationary ligands bonded to silica support.On ODS or TMS silicas, unlike on phenyl silica, aliphatic compounds gave slightly larger r-values than aromatic compounds, indicating that the shape of the ligand recognizes the hydrophobic surfaces of aliphatic and aromatic solute molecules. On TMS and phenyl silicas, the degree of solute hydrophobicity contributing to its retention is about 90% and 85% of that on ODS silica, respectively. On the other hand, on TMS and phenyl silicas, the polar functional group on the solute molecule brought about a smaller decrease in retention than on ODS silica.     

Reversed phase liquid chromatographic separation of some mono-substituted phenols with calix[6]arene-ρ-sulfonate-modified eluents

Summary The utility of water-soluble calix[6]arene--sulfonate as a mobile phase additive has been investigated for the reversed phase liquid chromatographic separation of some monosubstituted phenol isomers. Retention factors and separation factors for regioisomers of methoxyphenol, aminophenol, and nitrophenol were measured using methanol — water and acetonitrile —water mobile phases of varying composition containing calix[6]arene--sulfonate, and the values were compared with those obtained using mobile phases containing no additive. It was observed that addition of calix[6]arene--sulfonate to both acetonitrile — water and methanol —water caused a reduction in the retention of the phenol isomers but generally increased the separation between them, thereby improving the overall separation efficiency.     

Use of Unmodified and Aminated β-Cyclodextrins for the Separation of Enantiomers of Amino Acid Derivatives by High-Performance Liquid Chromatography

The separation of enantiomers of amino acid derivatives with modifiers of different structures on -cyclodextrin and aminated -cyclodextrin by reversed-phase high-performance liquid chromatography was studied. The dependence of the capacity factors of adsorbates on the concentration of the organic component in the mobile phase was examined. It was found that the retention of amino acid derivatives on unmodified -cyclodextrin and aminated -cyclodextrin increases with the hydrophobicity of the modifying agent of amino acid. In addition, for aminated -cyclodextrin, electrostatic interactions of ionized adsorbates and protonated surface amino groups substantially contribute to the retention. It was demonstrated that the recognition ability of aminated -cyclodextrin is affected by the structure of amino acid and its degree of dissociation.     

Comparison of normal-phase and reversed-phase gravity-flow column methods for provitamin A determination

Summary Considering the high cost of HPLC, the use of preparative C₁₈ packing material as stationary phase in classical gravity-flow columns is proposed as an alternative for developing countries. In the present study, three methods were compared, one utilizing a normal-phase MgO: HyfloSupercel column, developed with increasing concentration of acetone in petroleum ether and two utilizing C₁₈ reversed-phase columns developed with acetonitrile-methanol-chloroform (RP-C₁₈ method) or with decreasing concentration of water in acetone (modified RP-C₁₈ method). Percent recoveries of -carotene from the columns were 98, 96 and 98% and of the entire analysis (-carotene added to kale) 92, 89 and 92%, for the normal phase, RP-C₁₈ and modified RP-C₁₈ methods respectively. No significant difference was observed in the provitamin A contents (exclusively -carotene) of two leafy vegetables determined by the modified reversed-phase and normal-phase methods; however, significantly lower values were obtained by the RP-C₁₈ method. The situation became more complicated when samples containing other provitamins were analyzed. For squash both types of stationary phase could separate - and -carotene; however, for tomato and red-fleshed papaya, part of lycopene remained mixed with -carotene in both reversed-phase columns. Although -cryptoxanthin was separated in the modified RP column, it was also mixed with lycopene in the RP column. For all samples, the normal-phase column demonstrated much better separation.     

Direct separation of amino enantiomers by high performance ligand exchange chromatography on chemically bonded chiral phases

Summary Various chiral chemically bonded stationary phases for the separation of amino acid enantiomers by high performance ligand exchange chromatography are described. The phases were synthesized by treating silica gel with 3-glycidoxypropyltrimethoxysilane and bonding L-amino acids such as L-azetidine carboxylic acid, L-pipecolic acid or L-phenylalanine to the product. The best results were obtained with L-pipecolic acid as a fixed ligand. Nearly all common amino acid enantiomers could be separated.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Crosslinking of alkylpolysiloxane filsm on various types of glass surfaces including fused silica using γ-radiation of a60cobalt-source. Comparison to crosslinking by thermal peroxid treatment

Summary Crosslinking of alkylpolysiloxane stationary phases, especially in thick film capillary columns, is useful for the homogenous coating of fused silica and pretreated alkaliglass surfaces. The films of the stationary liquid are immobilized against solvent rinsing using CH₂Cl₂, pentane, and acetone, and maintain homogenity even at high temperature. Various doses of -radiation from a⁶⁰Co. source were used for the crosslinking instead of the thermal peroxid treatment recently described by other authors. The effect of the -radiation crosslinking-procedure was investigated in comparison to the peroxid method in regard of: decrease of stationary phase content by solvent rinsing, separation efficiency, tailing behaviour, and bleeding of the columns obtained. Similar results as with the cumylperoxid-treatment were achieved using the -radiation-method. By -radiation no polar functional groups or moleculs are introduced into the stationary phase, however, as with the peroxid method. Less than 20% of the various stationary liquids are usually removed from the columns by solvent rising after crosslinking using both methods depending on the doses of radiation and the cumylperoxid concentration applied respectively.Dedicated to Dr. L. S. Ettre on the occasion of his 60th birthday.     

Differential interactions of cyclodextrins with hydrophobic derivatives of sepharose CL-4B

The partition coefficients K_av between the solution phase and octyl- and phenyl-sepharose CL-4B were determined for three cyclodextrins (CDs), -CD, -CD and -CD in various aqueous solutions, as a measure of their interactions with the two hydrophobic ligands. K_av of the CDs increased in the order of -CD<-CD<-CD for octyl-sepharose CL-4B and -CD<-CD<-CD for phenyl-sepharose CL-4B. In all cases, K_av increased by increasing NaCl concentration in the aqueous solution phase and also by lowering temperature, but in the presence of NaBr and NaSCN, both chaotropic salts, K_av decreased markedly. The spontaneity of the transfer of the CDs from the aqueous solution phases to the gel phases was due to the enthalpy decrease. It was shown that discrete separation of the three CDs can be achieved by the hydrophobic chromatography on a short column (1×25cm) of octyl-sepharose CL-4B by adjusting the NaCl concentration and temperature.