Enantioselective HPLC separation of bioactive C5-chiral 2-pyrazolines on lux amylose-2 and lux cellulose-2: Comparative and mechanistic approaches

Stereoselective analytical HPLC separations have been developed for a series of biologically active chiral 2-pyrazolines (1-22) to be used in monitoring their resolution reactions or to custom semipreparative HPLC separations prior to biological assessment of both enantiomers. Polysaccharide-based chiral stationary phases (CSPs), namely, Lux amylose-2 and cellulose-2, have been used. Both normal (n-hexane/ethanol) and polar organic (ethanol, methanol, acetonitrile, or mixtures thereof) elution modes were very beneficial for the achievement of baseline separations. The impact of various chemical moieties embedded in the structures of 2-pyrazolines 1-22 and the adopted stationary phases on chiral recognition has been investigated. A case of reversed order of elution following alterations in either stationary phase or elution mode has been observed. Our findings recommend that normal elution mode can be used for optimizing semipreparative HPLC methods whereas polar organic mobile phases (such as acetonitrile and ethanol) are more suited to stereoselective reactions monitoring, routine quality control work, or for pharmacological and toxicological assays. These results settle the implementation of polysaccharide-based CSPs using different elution modes and declare the practicality of such CSPs in stereoselective HPLC.     

Determination of Polyphenylarenes and Polynuclear Aromatic Hydrocarbons by Reversed Phase HPLC

Abstract

Standard mixtures of polycyclic compounds are analyzed by reversed phase microcapillary liquid chromatography. Acetonitrile/water and acetonitrile/THF/water are employed as stationary phases, and capacity ratios are reported. Polyphenylarenes can be distinguished from polynuclear aromatic hydrocarbons by a large shift in capacity ratios when changing the mobile phase. The shift in capacity ratios is most significant for 1,3,5-triphenylbenzene, which is demonstrated by a gradient elution. The influence of THF and water on retention behaviour of solutes is described.     

Separation of Cyclodextrins Using Cyclodextrin Bonded Phases

Abstract

Two different cyclodextrin bonded phases (α and β) were used for the separation of α-, β-and γ-cyclodextrins. The β-cyclodextrin phase was found to be, in general, more effective at resolving the cyclodextrins than the α-cyclodextrin bonded phase. Acetonitrile/water mixtures were used as mobile phases. The effect of mobile phase composition on retention and resolution is examined. The elution order was found to be size dependent. The results are discussed in terms of the overall retention mechanism.     

HPLC Phospholipid Separation Mechanisms on Silica,Amino, and Diol Columns

Abstract

The HPLC separation of phospholipid mixtures was investigated on silica, amino, and diol columns, using mobile phases consisting of acetonitrile, methanol, and phosphoric or trifluoroacetic acids in varying proportions. An explanation of the mechanism of these separations is given with regard to the individual column types.     

Isolation by Means of Preparative Reversed-Phase Liquid Chromatography of Epimeric Alcohols Formed Upon Reduction of Pregnenolone and Progesterone

Abstract

A method is described which permits complete separation on a preparatory scale of the 20R and 20S epimeric alcohols obtained from lithium aluminium hydride and sodium borohydride reduction of pregnenolone and progesterone, respectively. The retention behaviour and resolution obtained on chromatography of the epimers on C-18 bonded phase material and elution with different acetonitrile/water and methanol/water mobile phases were studied. The order of retention is in both cases in accordance with ¹H-NMR chemical shift data which indicate a stable conformation with a more exposed 20-OH group in the 20S (=20α) epimer. Deviations from the elution order expected for true reversed-phase retention mechanisms were found on elution with mobile phase systems of reduced water content.     

The use of acetone as a substitute for acetonitrile in analysis of peptides by liquid chromatography/electrospray ionization mass spectrometry

The recent worldwide shortage of acetonitrile has prompted interest in alternative solvents for liquid chromatography/mass spectrometry (LC/MS). In this work, acetone was substituted for acetonitrile in the separation of a peptide mixture by reversed‐phase high‐performance liquid chromatography (RP‐HPLC) and in the positive electrospray ionization mass spectrometry (ESI‐MS) of individual peptides. On both C12 and C18 stationary phases, the substitution of acetone for acetonitrile as the organic component of the mobile phase did not alter the gradient elution order of a five‐peptide retention standard, but did increase peak width, shorten retention times, and increase peak tailing. Positive ESI mass spectra were obtained for angiotensin I, bradykinin, [Leu⁵]‐enkephalin, and somatostatin 14 dissolved in both acetonitrile/water/formic acid (25%/75%/0.1%) and acetone/water/formic acid (25%/75%/0.1%). Under optimized ESI‐MS conditions, the mass spectral response of [Leu⁵]‐enkephalin was increased two‐fold when the solvent contained acetone. The substitution of acetone for acetonitrile resulted in only slight changes in the responses of the remaining peptides. A higher capillary voltage was required for optimum response when acetone was used. Compared with acetonitrile/water/formic acid (50/50/0.1%), more interfering species below m/z = 140 were found in the ESI‐MS spectra of acetone/water/formic acid (50/50/0.1%). Copyright © 2009 John Wiley & Sons, Ltd.     

Enhanced‐fluidity liquid chromatography using mixed‐mode hydrophilic interaction liquid chromatography/strong cation‐exchange retention mechanisms

The potential of enhanced‐fluidity liquid chromatography, a subcritical chromatography technique, in mixed‐mode hydrophilic interaction/strong cation‐exchange separations is explored, using amino acids as analytes. The enhanced‐fluidity liquid mobile phases were prepared by adding liquefied CO₂ to methanol/water mixtures, which increases the diffusivity and decreases the viscosity of the mixture. The addition of CO₂ to methanol/water mixtures resulted in increased retention of the more polar amino acids. The “optimized” chromatographic performance (achieving baseline resolution of all amino acids in the shortest amount of time) of these methanol/water/CO₂ mixtures was compared to traditional acetonitrile/water and methanol/water liquid chromatography mobile phases. Methanol/water/CO₂ mixtures offered higher efficiencies and resolution of the ten amino acids relative to the methanol/water mobile phase, and decreased the required isocratic separation time by a factor of two relative to the acetonitrile/water mobile phase. Large differences in selectivity were also observed between the enhanced‐fluidity and traditional liquid mobile phases. A retention mechanism study was completed, that revealed the enhanced‐fluidity mobile phase separation was governed by a mixed‐mode retention mechanism of hydrophilic interaction/strong cation‐exchange. On the other hand, separations with acetonitrile/water and methanol/water mobile phases were strongly governed by only one retention mechanism, either hydrophilic interaction or strong cation exchange, respectively.     

High-Performance Liquid Chromatography Separations of Nitrosamines. IV. Effect of Temperature on the Separation of Nitrosamino Conformers

Abstract

The separation of the syn and anti conformers of a selected group of nitrosamines was achieved only after the temperature of the cyclodextrin bonded silica column was lowered to ?20°C. In certain cases it was apparent that a lower temperature is required for baseline separations. The mobile phases used were 90% acetonitrile/10% triethylammonium acetate, pH 5 for the nitrosamino acids, and tetrahydrofuran for the cyclic nitrosamino isomers.     

Application of reversed-phase high-performance liquid chromatography for the separation of deuterium and hydrogen analogs of aromatic hydrocarbons

Substitution of hydrogen by deuterium in aromatic hydrocarbons can alter various properties significantly. Benzene, toluene, naphthalene, anthracene, phenanthrene, biphenyl and durene are separated from their deuterium analogs by reversed-phase (C₁₈ liquid chromatography with acetonitrile/water mobile phases and ultraviolet detection. Deuterium compounds always elute before the hydrogen analog; possible explanations are given. The elution pattern and retention times of anthracene and phenanthrene were unchanged when D₂O replaced H₂O as the mobile phase component.     

Separating Ability of Some Polar Mobile Phases in Reverse Phase High Performance Liquid Chromatography

Abstract

Homologous series of n-alkanes, n-alcohols, ethers, esters, amides and ketones were studied as solutes. Methanol, acetonitrile, ethanol, dioxane, 2-propanol and tetrahydrofuran were mobile phases. Spherisorb ODS columns with light and heavy octadecyl coatings were used. Methanol and acetonitrile were found to be the most selective solvents for reverse phase separation. The polar amides, alcohols, and ketones behaved differently from nonpolar solutes in acetonitrile mobile phases. Results were interpreted with the solvophobic theory.     

Application of the Solvatic Model for Prediction of Retention in RP-LC for Multi-Step Gradient Profiles

Application of the solvatic retention model of reversed-phase liquid chromatography was studied to predict retention of phenylisothiocyanate derivatives of amino acids from structural formulae and stationary and mobile phase properties. The gradient elution mode with methanol and acetonitrile aqueous mobile phases was used. It was shown that practically acceptable prediction or retention time values can be achieved after the first approximation step when experimental data of one run are used. The zero approximation level predictions—from structural formulae, column and mobile phase properties can be used as a “first guess” method from which further optimization can begin.

     

Determination of Glutamine and Asparagine by Isocratic Elution Reverse Phase Liquid Chromatography with Fluorescent Detection

Abstract

A method was developed specifically for the determination of glutamine and asparagine in the presence or absence of other amino acids. The amino acids were derivatized by o-phthalaldehyde/ 2-mercaptoethanol and separated by isocratic elution with a mobile phase consisting of acetonitrile and sodium acetate buffer. An application of the method for the analysis of glutamine and asparagine in the enzymatic hydrolysate of cottonseed protein is described.     

Liquid Chromatographic Behavior of Nitrogen Compounds

Abstract

Chromatographic behavior of nitrogen compounds differed from others. The column efficiency was poor for the compounds and sometimes solutes were not eluted out from a column. Therefore, the elution volume of alkylamines, anilines, pyridines, pyrazines, quinolines and aminopolyaromatic hydrocarbons was measured on a methacrylate gel and octadecyl bonded silica gels in pH controlled acetonitrile/water mixtures. The solvent effect on the dissociation constant differs from that obtained for aromatic acids. The values in acetonitrile/water mixtures are smaller than those obtained in 100% water. The linear relation between log P and log k′ values is obtained in eluents of pH 7 where the retention of these compounds is maximized. Some hydrophobic fragmental constants are proposed from this result. Prediction of retention time of these compounds from their log P values can be done in the individual groups on octadecyl bonded silica gels in pH controlled acetonitrile/water mixtures.     

A Comparison of Radially-Compressed and Stainless Steel Columns for the Reversed-Phase Ion-Pair Separation of Phencyclidine Synthetic Mixtures

Abstract

The reversed-phase ion-pair HPLC separation of phencyclidine synthetic mixtures was optimized utilizing Radial-Pak radially compressed columns. Variables examined in the optimization included column type (C-18, C-8, or CN), pairing ion (methane-, pentane-, hexane-, or octane sulfonates) and mobile phase composition (varying concentrations of methanol or acetonitrile in water). The chromatographic behavior of the phencyclidine mixtures in the various systems utilizing radially compressed columns is compared and contrasted to a similar previous study which examined similar variables on stainless steel columns. The optimum system for radially compressed columns was found to consist of a Radial-Pak C-18 column and a mobile phase of 85:15 MeOH:H₂O, 2.5% acetic acid, 1% triethylamine and 5mM sodium hexane sulfonate.     

Evaluation of ternary mobile phases for reversed-phase liquid chromatography: Effect of composition on retention mechanism

The effect of varying mobile phase composition across a ternary space between two binary compositions is examined, on four different reversed-phase stationary phases. Examined stationary phases included endcapped C8 and C18, as well as a phenyl phase and a C18 phase with an embedded polar group (EPG). Mobile phases consisting of 50% water and various fractions of methanol and acetonitrile were evaluated. Retention thermodynamics are assessed via use of the van’t Hoff relationship, and retention mechanism is characterized via LSER analysis, as mobile phase composition was varied from 50/50/0 water/methanol/acetonitrile to 50/0/50 water/methanol acetonitrile. As expected, as the fraction of acetonitrile increases in the mobile phase, retention decreases. In most cases, the driving force for this decrease in retention is a reduction of the enthalpic contribution to retention. The entropic contribution to retention actually increases with acetonitrile content, but not enough to overcome the reduction in the enthalpic contribution. In a similar fashion, as methanol is replaced with acetonitrile, the v, e, and a LSER system constants change to favor elution, while the s and c constants change to favor retention. The b system constant did not show a monotonic change with mobile phase composition. Overall changes in retention across the mobile phase composition range varied, based on the identity of the stationary phase and the composition of the mobile phase.     

Measurement of the elution strength and peak shape enhancement at increasing modifier concentration and temperature in RPLC

Two approaches are proposed to measure the effect of different experimental factors (such as the modifier concentration and temperature) on the elution strength and peak shape in reversed-phase liquid chromatography, which quantify the percentage change in the retention factor and peak width (referred to the weakest conditions) per unit change in the experimental factor. The approaches were applied to the separation of a set of flavonoids with aqueous micellar mobile phases of the surfactant Brij-35 (polyoxyethylene(23)dodecanol), in comparison with acetonitrile–water mixtures, using an Eclipse XDB-C18 column. The particular interaction of each flavonoid with the oxyethylene chains of Brij-35 molecules (adsorbed on the stationary phase or forming micelles) changed the elution window, distribution of chromatographic peaks and partitioning kinetics, depending on the hydroxyl substitution in the aromatic rings of flavonoids. At 25?°C, peak shape with Brij-35 mobile phases was significantly poorer with regard to acetonitrile–water mixtures. At increasing temperature, the efficiency of Brij-35 increased, approaching at 80?°C the values obtained at equilibrium conditions, already reached with acetonitrile at 25?°C.     

Separation of fluorescent 1,N6 ethenoderivatives of diadenosine polyphosphates and their enzymatic degradation products by reversed-phase high-performance liquid chromatography

Summary The retention, selectivity and elution order of fluorescent 1,N⁶-etheno derivatives of diadenosine polyphosphates and their enzymatic degradation products on octadecyl and phenyl-bonded silica columns have been studied as a function of mobile phase pH, ionic strength and organic modifier content. Good separations of the compounds of interest were achieved using mobile phases of around 0.1M potassium phosphate buffers at neutral pH containing approximately 10% methanol or 4% acetonitrile for C₁₈ columns and 5% methanol or 1.5% acetonitrile for phenyl columns. The data obtained were used to establish isocratic assays for diadenosine polyphosphate cleaving activities from chromaffin cells using Di(1,N⁶-ethenoadenosine) polyphosphates as fluorogenic substrate analogues followed by fluorescence detection.     

Preparation and separation of mixed-ligand Fe(II) complexes containing 1,10-phenanthroline-5,6-dione as ligand

The synthesis, separation, and characterization of mixed-ligand iron(II) complexes containing 1,10-phenanthroline (phen), 1,10-phenanthroline-5,6-dione (pdon), and NCS^? are reported. The mixed-ligand complexes [Fe(phen)(pdon)₂]²⁺ and [Fe(phen)₂(pdon)]²⁺ were prepared from iron(II) sulfate hepta hydrate and both ligands. The mixture of both complexes formed regardless the ratio of the ligands or the reaction time; therefore, the complexes were separated successfully on the reversed phase (RP) Develosil RP-Aqueous [C30] 5?µm, 150?×?4.6?mm column by two different methods. The first method was the ion paired RP chromatography performed under gradient elution with acetonitrile–water containing 0.001?mol?L^?1 KPF₆ aqueous as mobile phases. The second method was the RP chromatography performed under gradient elution with methanol and water as mobile phases. The gradient elution with water–methanol as eluents was preferred for the semi preparative separations allowing one to use the complexes without further purification upon separation, different than the first method and its variations so far. Three complexes (5, 6, and 7) were characterized by electrospray ionization mass spectrometry, NMR, UV-Vis, and IR.     

Enantioseparation of Chiral Antimycotic Drugs by HPLC with Polysaccharide-Based Chiral Columns and Polar Organic Mobile Phases with Emphasis on Enantiomer Elution Order

The separation of enantiomers of 10 chiral antimycotic drugs was studied on polysaccharide-based chiral columns with polar organic mobile phases. The emphasis was placed on some interesting examples of enantiomer elution order reversal observed depending on the chemistry of the chiral selector, separation temperature, major component, as well as the minor additive in the mobile phase. In particular, it was found that the elution order of enantiomers of chiral drug terconazole was opposite on cellulose- and amylose-based columns with the same pendant group. The affinity pattern of enantiomers of another chiral drug bifonazole was opposite towards to two amylose-based chiral selectors with different pendant groups. The affinity pattern of terconazole enantiomers also changed on some columns when the alcohol-based mobile phase was replaced with acetonitrile. An interesting effect of the minor acidic (formic acid) additives to the mobile phase on the affinity pattern of terconazole enantiomers was observed on Cellulose-2 and Cellulose-4 columns. In addition, a reversal of elution order of bifonazole enantiomers was observed on Amylose-2 column by variation of a separation temperature.

     

Analysis of polar peptides using a silica hydride column and high aqueous content mobile phases

The retention behavior of a set of polar peptides separated on a silica hydride stationary phase was examined with a capillary HPLC system coupled to ESI‐MS detection. The mobile phases consisted of formic acid or acetic acid/acetonitrile/water mixtures with the acetonitrile content ranging from 5 to 80% v/v. The effects on peptide retention of these two acidic buffer additives and their concentrations in the mobile phase were systematically investigated. Strong retention of the peptides on the silica hydride phase was observed with relatively high‐organic low‐aqueous mobile phases (i.e. under aqueous normal‐phase conditions). However, when low concentrations of acetic acid were employed as the buffer additive, strong retention of the peptides was also observed even when high aqueous content mobile phases were employed. This unique feature of the stationary phase therefore provides an opportunity for chromatographic analysis of polar peptides with water‐rich eluents, a feature usually not feasible with traditional RP sorbents, and thus under conditions more compatible with analytical green chemistry criteria. In addition, both isocratic and gradient elution procedures can be employed to optimize peptide separations with excellent reproducibility and resolution under these high aqueous mobile phase conditions with this silica hydride stationary phase.