Reversed phase liquid chromatography of alkyl-imidazolium ionic liquids

Eleven 1-alkyl-3-methyl imidazolium ionic liquid (IL) salts were analyzed in reversed phase mode with a Kromasil C18 column. The mobile phases were water-rich acetonitrile solutions (water content > or =70%, v/v) without any added salts. It is shown that it is possible to separate different ILs sharing the same cation and differing by the anion when salt-free mobile phases are used. When a buffer, acetate or phosphate salt, or any salt, such as sodium chloride or sodium tetrafluorobarate, is added to the mobile phase, the ILs differing only by their anions cannot be separated. ILs with different alkyl chains in the imidazolium cation are separated by mobile phases with or without added salts following a hydrophobic interaction behavior: log k is proportional to nC, the carbon number of the alkyl chain. Important differences in ion/stationary phase interactions are observed depending on the ionic content of the mobile phase. With salt-free mobile phases, the IL/C18 stationary phase interactions correspond to concave isotherms associated with fronting peaks for all ILs. With mobile phase containing 0.01 M of salt, tailing IL peaks correspond to convex adsorption isotherms. Also, the IL retention factor depends on the concentration and nature of the added salt. Hexafluorophosphate chaotropic anions can adsorb on the Kromasil C18 surface dramatically increasing the imidazolium cation retention factors.     

Comparison of Reversed Stationary Phases for the Chromatographic Separation of Inorganic Analytes Using Hydrophobic Ion Mobile Phase Additives

Abstract

Alkyl-modified silica (RSi) and polystyrenedivinylbenzene (PRP-1) stationary phases are compared for the chromatographic separation of inorganic analyte anions and cations using hydro-phobic ions of opposite charge as mobile phase additives. Tetra-alkylammonium salts were used for anion separations and alkyl sulfonate salts for cation separations. Two major equilibria influence the retention of analyte ions on PRP-1. These are: retention of the hydrophobic ion on PRP-1 and an ion exchange selectivity between the hydrophobic counterion and the analyte ion. When using RSi retention is also influenced by ion exchange at residual silanol groups, which act as weak cation exchange sites. Mobile and stationary phase variables that influence analyte retention are identified. Optimization of these provides favorable eluting conditions for the separation of inorganic ionic analytes. Of particular interest is the potential use of PRP-1 and RSi columns for the separation of inorganic cations; conditions for the separation of alkali metals and alkaline earths are discussed.     

Comparison and evaluation of HIC columns of different hydrophobicity

Summary Retention and selectivity in hydrophobic interaction chromatography (HIC) depend both on the type of stationary phase and on the mobile phase. In the last few years various high performance packing materials and columns have been introduced for HIC resulting in a range of different retentions and selectivity. We have investigated the effect of the stationary phase on the retention of various proteins. The retention of some solutes of different hydrophobicities were measured on three commercial HIC columns (TSK-Phenyl, Synchropack-Propyl, CAA-HIC) under isocratic conditions using water-methanol mixtures as eluent. The log k_w values determined according to the literature were devalues determined according to the literature were dependent on the type and structure of the stationary phase and indicated a much less hydrophobic character for these columns than that obtained for reversed phase columns. Gradient separations were then carried out on a standard protein mixture using ammonium sulfate and sodium citrate to change the gradient time. In order to compare the effect of the stationary phase and the two salts investigated apparent capacity factors (k_g) were determined and plotted against the gradient time obtained for the three columns in the two eluent system. It was shown that the type of stationary phase had a significant effect on the retention of proteins. In addition, the effect of the mobile phase composition, i.e. salt type, was considerably different on the various stationary phases. In order to exploit the potential of HIC to modulate selectivity for the separation of proteins, the combined effect of the stationary phase and the type of salt should be taken into account.Dedicated to Professor Leslie S. Ettre on the occasion of his 70th birthday.     

Separation of Free Amiimo Acids on Reverse Stationary Phases Using an Alkyl Sulfonate Salt as a Mobile Phase Additive

Abstract

Alkylsulfonate (RSO₃ ^?) salts were evaluated as mobile phase additives for the separation of free amino acids on reverse stationary phases using an acidic mobile phase where the amino acids are cations. The enhanced amino acid retention is the result of two major interactions, one being retention of the RSO₃ ^? salt on the stationary phase and the other an ion exchange selectivity between the amino acid analyte cation and the RSO₃ ^? countercation, or other countercations in the mobile phase. Major mobile phase variables are: type and concentration of RSO₃ ^? salt (the studies focused on C₈SO₃ ^? salts), presence of organic modifier, type of countercation present, and mobile phase pH and ionic strength. Alkyl modified silica and polystyrenedivinyl-benzene copolymeric reverse stationary phases were compared. A mobile phase gradient, increasing per cent organic modifier was shown to be best, is necessary for separating complex mixtures of polar and nonpolar or basic amino acids. The procedure is applicable to the identification and/or determination of amino acids in mixtures or in peptides after hydrolysis.     

Reversed-phase liquid chromatography analysis of alkyl-imidazolium ionic liquids II. Effects of different added salts and stationary phase influence

Two mixtures of four 1-alkyl-3-methylimidazolium ionic liquids (ILs) salts associated to the anions tetrafluoroborate or hexafluorophosphate were analyzed by reversed-phase liquid chromatography with three different stationary phases: Kromasil C(8), Zorbax Extend C(18) and Zorbax Sb-Aq. The effect on retention of various inorganic salts (NaCl, NaH(2)PO(4,) NaBF(4), NaClO(4) and NaPF(6)) added to acetonitrile/water mobile phases was studied. The three columns gave similar separation profiles. In all cases, the retention of ILs increased with the increasing affinity of the inorganic anions for the apolar stationary phases; a phenomenon called chaotropicity. The chaotropic anion order is Cl(-) approximately H(2)PO(4)(-) < BF(4)(-) approximately ClO(4)(-) < PF(6)(-). It is established that the presence of chaotropic anions in the mobile phase do not permit to differentiate between ILs associated to different anions. However, chloride or dihydrogenphosphate added salts do not fully screen the retention differences between ILs associated with different anions. Distorted and even split peaks may appear in the chromatogram depending on the nature and concentration of the injected ILs. In the RPLC analysis of imidazolium-based IL, it is recommended to add to the mobile phase significant amounts of a salt containing a chaotropic anion. This salt addition will improve the IL peak shapes and give reproducible retention factors. LODs in the low microgram range ( approximately 5 nmol) were obtained with the Kromasil C(8) column with a 50/50 acetonitrile-water mobile phase containing 0.01 M NaPF(6) added salt and 230 nm UV detection.     

The Separation of Organic Analyte Cations on a Low-Capacity Cation Exchange Column Using Indirect UV Detection

Abstract

The retention of organic analyte cations on a low-capacity cation exchange column using indirect UV detection was studied. It was found that a combination of cation exchange/reversed-phase interactions affected the retention of organic analyte cations provided the analytes have both a cationic charge site and a hydrophobic center. The factors that influenced the organic analyte cation retention were: concentration of organic modifier, concentration of UV absorbing analyte, pH, and mobile phase ionic strength. Elution orders for several of the organic analytes studied on the low-capacity cation exchange column were different than those observed on silica-based strong cation exchange columns.     

Study of the selectivity,retention mechanisms and performance of alternative silica-based stationary phases for separation of ionised solutes in hydrophilic interaction chromatography

The separation of a mixture of neutral, strongly acidic and strongly basic compounds was studied in hydrophilic interaction chromatography using a bare silica phase, and bonded silica phases with diol, zwitterionic, amide and hydrophilic/hydrophobic groups. The mobile phase was acetonitrile–ammonium formate buffer at low pH. Differences in selectivity between these various columns indicate that the stationary phase cannot function merely as an inert support for a water layer into which the solutes partition from the bulk mobile phase. Attempts to fit the retention data to equations which describe either partition or adsorption mechanisms were inconclusive. Ion exchange was a significant contributor to the retention of ionised bases on all columns studied. Van Deemter plots indicated that the efficiency as a function of flow rate varied between the columns, which might be attributable in part to the presence of either monomeric or polymeric bonded phase layers.     

Effect of chemically bonded stationary phases and mobile phase composition on beta-blockers retention in RP-HPLC

The effects of stationary and mobile phase on retention of 18 beta-adrenolytic drugs (beta-blockers) have been studied. Four 'deactivated surface' stationary phases (polar-embedded or end-capped) were examined. Special attention was drawn to the cholesterolic (SG-CHOL) and alkylamide (SG-AP) stationary phases, and their application for analysis of the compounds. The retention of analyzed substances was also examined in terms of mobile phase composition. Sixteen different configurations of mobile phases were prepared, all based on methanol and acetonitrile with ammonium acetate and ammonium formate. The difference in retention between ammonium formate and acetate water solutions, and peak shape changes related to the addition of triethylamine (TEA), were investigated. Principal component analysis was used to find the similarities between stationary phases. Polar-embedded phases synthesized on the same sorbent possess very similar properties. All phases based on silica gel compared with the monolithic column also showed similarities in retention of beta-blockers. The addition of TEA to the mobile phase did not influence strongly the retention, and analysis of asymmetry factors showed only a little peak broadening for a few compounds on the monolithic column.     

单柱离子色谱法测定一价阳离子的流动相研究

对单柱离子色谱法测定一价阳离子的流动相进行了系统研究，阐述了一价阳离子的保留行为和电导检测行为与流动相之间的关系，分别对无机酸（硝酸）、有机酸（柠檬酸）和芳香碱（苯胺）为流动相测定一价阳离子进行了讨论，其中有机酸和无机酸是较为适宜的流动相。     

Micellar bile salt mobile phases for the liquid chromatographic separation of routine compounds and optical, geometrical, and structural isomers

Aqueous solutions of bile salts, i.e. sodium cholate (NaC), sodium deoxycholate (NaDC), and sodium taurocholate (NaTC), are characterized and evaluated as reversed-phase liquid chromatographic (RPLC) mobile phases. The separation of the ASTM-recommended RPLC test mix in addition to more than 50 other compounds on a C18 column demonstrates the viability of these bile salts as HPLC mobile phases. The Armstrong-Nome theory was applied and found to adequately describe the partitioning behavior of solutes eluted with these bile salts at low surfactant concentrations. The effect of alcohol additives on chromatographic retention and efficiency was also assessed. Not only are the bile salt molecules rigid and chiral, but they form helical micellar aggregates as well. Consequently, many isomeric compounds can be easily resolved with this mobile phase additive. The base-line resolution of some binaphthyl-type enantiomers with a standard C18 column and the bile salt micellar mobile phases is also demonstrated. In addition, these bile salt mobile phases may be preferable to conventional hydroorganic mobile phase systems for the separation of many classes of routine compounds. A brief prospectus on the future utilization of bile salts in liquid chromatography is presented.     

Evaluation of the influence of residual silanol groups on the total retention in reversed-phase chromatography

It was demonstrated that the characteristics of stationary phases for reversed-phase chromatography can be compared by analyzing the retention of several compounds belonging to a homologous series during elution with one and the same composition of mobile phase or the retention of a nonpolar compound during elution with several mobile phases of different compositions. The slope of the corresponding linear correlations can be used to characterize the hydrophobicity of the columns (stationary phases) under study. To analyze the polar interactions between the sorbate and the stationary phase it is advantageous to study the retention of a polar substance in several (at least in two) mobile phases of different compositions. The degree of displacement of the linear correlation relative to that for nonpolar substances can be considered a measure of the influence of residual silanol groups on the total retention characteristics.     

Complexation and chromatographic studies involving water soluble calixarenes and nitrophenols

Summary In this study, an investigation of the utilisation of the sodium salts of 4-sulphonic calix[4]arene and 4-sulphonic calix[6]arene as mobile phase additives in reversed phase HPLC is described. Consistent with earlier work these mobile phase additives were shown to strongly influence the retention behaviour and separation factor of 2-, 3- and 4-nitrophenols. Proton NMR and UV spectroscopic data is presented to demonstrate that this chromatographic behaviour is due to pH effects and not due to complexation between the nitrophenols and the calixarenes.     

Hydrophilic interaction liquid chromatography in the separation of a moderately lipophilic drug from its highly polar metabolites--the cardioprotectant dexrazoxane as a model case

This paper presents a systematic study of the retention behavior of a model bisdioxopiperazine drug, dexrazoxane (DEX) and its three polar metabolites (two single open-ring intermediates-B and C and an EDTA-like active compound ADR-925) on different stationary phases intended for hydrophilic interaction liquid chromatography (HILIC). The main aim was to estimate advantages and limitations of HILIC in the simultaneous analysis of a moderately lipophilic parent drug and its highly polar metabolites, including positional isomers, under MS compatible conditions. The study involved two bare silica columns (Ascentic Express HILIC, Atlantis HILIC) and two stationary phases with distinct zwitterionic properties (Obelisc N and ZIC HILIC). The chromatographic conditions (mobile phase strength and pH, column temperature) were systematically modified to assess their impact on retention and separation of the studied compounds. It was found that the bare silica phases were unable to separate the positional isomers (intermediates B and C), whereas both columns with zwitterionic properties (Obelisc N and ZIC HILIC) were able to separate these structurally very similar compounds. However, only ZIC HILIC phase allowed appropriate separation of DEX and all its metabolites to a base line within a single run. A mobile phase composed of a mixture of ammonium formate (0.5 mM) and acetonitrile (25:75, v/v) was suggested as optimal for the simultaneous analysis of DEX and its metabolites on ZIC HILIC. Thereafter, HILIC-LC-MS analysis of DEX and all its metabolites was performed for the first time to obtain basic data about the applicability of the suggested chromatographic conditions. Hence, this study demonstrates that HILIC could be a viable solution for the challenging analysis of moderately polar parent drug along with its highly polar metabolites including the ability to separate structurally very similar compounds, such as positional isomers.     

Reversed-phase high-performance liquid chromatography of amphoteric beta-lactam antibiotics: effects of columns, ion-pairing reagents and mobile phase pH on their retention times

The separation of five amino beta-lactam antibiotics by reversed-phase high-performance liquid chromatography was studied as an insight into their retention behaviour. These five amphoteric compounds are cephradine, cephalexine, cefaclor, ampicillin and amoxicillin. Both octadecylsilane-bonded silica (C18) columns and phenyl-bonded silica (phenyl) columns were used, with mobile phase pH values between 2.5 and 7.4. In the absence of ion-pairing reagents the retention times for all the five compounds were the shortest at pH 4-6. The phenyl column was found to improve the separation between cephradine and ampicillin at pH values lower than 3, when these two compounds appeared as fused peaks on the C18 on C18 columns, with mobile phases both with and without ion-pairing reagents, were compared. The addition of 0.005 or 0.02 M tetraethylammonium acetate to the mobile phase did not result in significant ion-pair formation, except at pH values higher than 5.5. A strong ion-pairing effect was obtained at pH values higher than 6 with 0.005 or 0.02 M tetrabutylammonium phosphate, and the retention was decreased at pH values lower than 4. On the other hand, 0.005 M heptanesulphonic acid exhibited an ion-pair retention effect at pH values lower than 5. The molecular structures and pK(a) values were used to account for the retention behaviour of these antibiotics in the various mobile phases.     

Mobile phase viscosity and velocity dependence on protein retention using nonequilibrium chromatographic techniques

Nonequilibrium chromatography (NEC) is an alternative chromatographic procedure for the separation of macromolecules. The retardation of a protein series is studied using a phosphate buffer as a mobile phase with various concentrations of glycerol fraction (used as a viscosity modifier) at different mobile phase velocities and a C1 column with a very low packing particle diameter as a stationary phase. It is shown that the two factors (viscosity and velocity) of the mobile phase constituted important parameters in the retention mechanism of the proteins in NEC. The retardation velocity domain is divided into two regions. For low velocity regions, the protein retention decreased with a mobile phase velocity increase. This retention is enhanced above a critical value of the mobile phase velocity. The transition between the two well-known NEC methods, slalom chromatography and hydrodynamic chromatography, is clearly visualized for the first time for the protein retention of particular values of the mobile phase velocity.     

Peculiarities of Mobile Phases Containing Formic Acid

Formic acid is often used for pH adjustment in RP–LC mobile phases. However, under certain circumstances, formic acid can also behave as an organic co-solvent. This study illustrates two scenarios when the effects of formic acid as co-solvent will be most noticeable. One scenario is for water-rich mobile phases, in which the addition of formic acid will cause small but significant decreases in retention of neutral analytes. The other scenario is in gradient chromatography. It is demonstrated that, during acetonitrile solvent gradients, desorption of formic acid from the column stationary phase occurs simultaneously, potentially causing a spike in UV detector baselines at the beginning of gradients. A pK _a value of formic acid obtained in 50% acetonitrile is also reported and the impact of organic solvent on the dissociation of formic acid is discussed.     

Relevance of pi-pi and dipole-dipole interactions for retention on cyano and phenyl columns in reversed-phase liquid chromatography

Previous work suggests that pi-pi interactions between certain solutes and both phenyl and cyano columns can contribute to sample retention and the selectivity of these two column types versus alkylsilica columns. Recent studies also suggest that dipole-dipole interactions are generally unimportant for retention on cyano columns. The present study presents data for 44 solutes, three columns and two different mobile phases that were selected to further test these conclusions. We find that pi-pi interactions can contribute to retention on both cyano and phenyl columns, while dipole-dipole interactions are likely to be significant for the retention of polar aliphatic solutes on cyano columns. When acetonitrile/water mobile phases are used, both pi-pi and dipole-dipole interactions are suppressed, compared to the use of methanol/water.     

Ultra-high-performance liquid chromatography fingerprinting method for chemical screening of metabolites in cultivation broth

A fingerprinting method for chemical screening of microbial metabolites, potential antibiotics, in spent cultivation broths is described. The method is based on high-throughput ultra-high-performance liquid chromatography (UHPLC) separation with UV detection (photodiode array detector). Thirteen antibiotic standards and four cultivation broths were used for the method development. The comparison of ten liquid–liquid and solid phase extraction protocols for sample clean-up and pre-concentration revealed that Oasis HLB C18 sorbent gives the best recoveries. The Acquity BEH C18 chromatographic column was chosen for the samples separation with respect to its universality, selectivity, efficiency and robustness. The method is presented by two 3D fingerprints for every sample that was obtained under different, acidic and alkaline, UHPLC conditions. The acidic mobile phase consisted of 0.5% phosphoric acid with methanol and the alkaline mobile phase of 1 mM ammonium formate, pH 9 with acetonitrile. Each pair of 3D fingerprints includes the following physico-chemical information: polarity (retention time), presence and characterization of chromophores (UV spectra), compound concentration (detector response), and acid–base properties (influence of different pH of the aqueous parts of mobile phases on retention times). The sample extraction and method validation were assessed with relative standard deviation (RSD) of 0.5, 5.0 and 20.0% for retention times, peak areas and minor compound peak areas, respectively.     

Effect of column temperature on enantioseparation of dihydropyrimidinones using alcohol solvated Chiralpak AS and AS-H columns

In this study we report the application of column temperature programs to compare the chromatographic behaviors of four commercially available dihydropyrimidinone (DHP) compounds on Chiralpak AS and AS-H phases under different alcohol solvation conditions such as 1-propanol (1-PrOH), 2-methyl-1-PrOH (2-Me-1-PrOH), 1-butanol (1-BuOH), 2-BuOH, and tert-BuOH (t-BuOH) in n-hexane (n-Hex). It was observed that the apparent retention factors of the DHP compounds on the AS phase in t-BuOH/n-Hex mobile phase had the largest reduction (>27%) among the mobile phases studied after a heating and cooling temperature cycle (10 to 50 back to 10 degrees C). However, the average reduction of the apparent retention factors of the compounds on t-BuOH solvated AS-H was less than 4%. No clear trend of changes in the apparent retention factors on AS and AS-H columns was observed in other alcohol modified mobile phases. Step-temperature programs showed that alcohol solvated AS phase had larger changes in the apparent retention factors with time than those of AS-H phase at the elevated temperature step (50 degrees C). Both t-BuOH solvated AS and AS-H phases showed kinetic behaviors in chromatographic processes at the elevated temperature.