Effect of chromatographic conditions on the separation and system efficiency for HPLC of selected alkaloids on different stationary phases

Retention parameters of alkaloid standards were determined on different stationary phases, i.e., octadecyl silica, base-deactivated octadecyl silica, cyanopropyl silica, preconditioned cyanopropyl silica, and pentafluorophenyl, using different aqueous eluant systems: acetonitrile-water mixtures; buffered aqueous mobile phases at pH 3 or 7.8; and aqueous eluants containing ion-pairing reagents (octane-1-sulfonic acid sodium salt and pentane-1-sulfonic acid sodium salt) or silanol blockers (tetrabutyl ammonium chloride and diethylamine). Improved peak symmetry and separation selectivity for basic solutes was observed when basic buffer, ion-pairing reagents, and, especially, silanol blockers as mobile phase additives were applied. The best separation selectivity and most symmetric peaks for the investigated alkaloids were obtained in systems containing diethylamine in the mobile phase. The influence of acetonitrile concentration and kind and concentration of ion-pairing reagents or silanol blockers on retention, peak symmetry, and system efficiency was also examined. The most efficient and selective systems were used for separation of the investigated alkaloids and analysis of Fumaria officinalis and Glaucium flavum plant extracts.     

The role of the dual nature of ionic liquids in the reversed-phase liquid chromatographic separation of basic drugs

The cationic nature of basic drugs gives rise to broad asymmetrical chromatographic peaks with conventional C18 columns and hydro-organic mixtures, due to the ionic interaction of the positively charged solutes with the free silanol groups on the alkyl-bonded reversed-phase packing. Ionic liquids (ILs) have recently attracted some attention to reduce this undesirable silanol activity. ILs are dual modifiers (with a cationic and anionic character), which means that both cation and anion can be adsorbed on the stationary phase, giving rise to interesting interactions with the anionic free silanols and the cationic basic drugs. A comparative study of the performance of four imidazolium-based ILs as modifiers of the chromatographic behaviour of a group of β-blockers is shown. The ILs differed in the adsorption capability of the cation and anion on C18 columns. Mobile phases without additive and containing a cationic (triethylamine, TEA) or anionic (sodium dodecyl sulphate, SDS) additive were used as references for the interpretation of the behaviours. The changes in the nature of the chromatographic system, at increasing concentration of the additives, were followed based on the changes in retention and peak shape of the β-blockers. The silanol suppressing potency of the additives, and the association constants between the solutes and modified stationary phase or additive in the mobile phase, were estimated. The study revealed that SDS and the ionic liquid 1-hexyl-3-methylimidazolium tetrafluoroborate are the best enhancers of chromatographic peak shape among those studied.     

On the use of ionic liquids as mobile phase additives in high-performance liquid chromatography. A review

The popularity of ionic liquids (ILs) has grown during the last decades in several analytical separation techniques. Consequently, the number of reports devoted to the applications of ILs is still increasing. This review is focused on the use of ILs (mainly imidazolium-based associated to chloride and tetrafluoroborate) as mobile phase additives in high-performance liquid chromatography (HPLC). In this approach, ILs just function as salts, but keep several kinds of intermolecular interactions, which are useful for chromatographic separations. Both cation and anion can be adsorbed on the stationary phase, creating a bilayer. This gives rise to hydrophobic, electrostatic and other specific interactions with the stationary phase and solutes, which modify the retention behaviour and peak shape. This review updates the advances in this field, with emphasis on topics not always deeply considered in the literature, such as the mechanisms of retention, the estimation of the suppressing potency of silanols, modelling and optimisation of the chromatographic performance, and the comparison with other additives traditionally used to avoid the silanol problem.     

Utility of ionic liquids in analytical separations

Ionic liquids (ILs), as separation media, have made significant contributions in the past decades in advancing research in gas chromatography (GC), liquid chromatography (LC), and capillary electrophoresis (CE). This review, covering reports published from the mid 1980s to early 2007, shows how ILs have been used so far in separation science, originally primarily as GC stationary phases and later as mobile phase additives (both millimolar and major percent levels) for LC and CE. Representative GC and LC chromatograms as well as CE electropherograms are shown. In addition, the very recent findings on the development of ionic liquids with surfactant properties and its applications for chiral and achiral analysis are discussed.     

Mobile-phase pH influence on the retention of some benzoic acid derivatives in reversed-phase chromatography

Five end-capped octadecyl RP stationary phases, among which one was a polar embedded stationary phase, were tested for the analysis of benzoic acid derivatives using two mobile phases with or without addition of formic acid (water pH was measured by a common approach; pH of water with addition of formic acid was 3.0 and without formic acid 5.8). The influence of mobile-phase pH on the retention of benzoic acid derivatives was under study. Consequently, Purospher-STAR and Alltima columns provided symmetrical peaks for benzoic acid derivatives at pH 3.0 and also at pH 5.8. Reprosil and Symmetry stationary phases showed poor peak shapes at higher pH of the mobile phase. Differences between the tested columns may be caused by surface heterogeneity. Another reason may be the presence of some atoms creating additional adsorption sites on the surface of Reprosil and Symmetry stationary phases. This can lead to enhanced silanol activity resulting in peak tailing. The addition of formic acid into the mobile phase improved peak shapes. The polar embedded C18 stationary-phase Synergi-Fusion-RP appeared as not a suitable column for the analysis of benzoic acid derivatives. Synergi-Fusion-RP provided asymmetrical peaks even if formic acid was added into the mobile phase.     

Comparison of aqueous and non-aqueous capillary electrochromatography for the separation of basic solutes

The analysis of basic compounds by capillary electrochromatography (CEC) on silica-based materials using conventional HPLC stationary phases has failed to address the problem of severe peak tailing and non-reproducible chromatography. Several new generation stationary phases were evaluated using aqueous and non-aqueous mobile phases. The best results were obtained in the aqueous mode using Waters Symmetry Shield RP-8, a material in which the residual silanol groups were shielded by an octylcarbamate function. For comparison, experiments were carried out using unmodified silica.     

A study of the behaviour of some new column materials in the chromatographic analysis of Cinchona alkaloids

Summary A major problem in the HPLC analysis of alkaloids is the poor peak shape and consequently low resolution, due to the interactions of the basic alkaloids with the residual acidic silanol groups of most reversed phase materials. The performance of new packing materials specially designed for the separation of basic compounds has been studied using mobile phases without the special additives commonly applied in the analysis of alkaloids. Strongly basic Cinchona alkaloids were used as test compounds. Retention characteristics and selectivities of each material were studied, after mobile phase optimisation for the column. The influence of the major factors (nature and content of the organic modifier, pH value, salt concentration) affecting resolution was studied. The mobile phases were chosen so that they could be used in thermospray LC-MS. The addition of salts to the mobile phase improves separation but in general the modification of the mobile phase gave little change in selectivity. The performance of silica-based C₁₈ material proved superior to the polymer materials tested.     

Suitability of ionic liquids as mobile‐phase additives in HPLC with fluorescence and UV detection for the determination of heterocyclic aromatic amines

The effects of several ionic liquids (ILs) as mobile‐phase additives in HPLC with fluorescence and UV–Vis detection for the determination of six heterocyclic aromatic amines were evaluated using two different C₁₈ stationary phases with moderate silanol activity. The studied ILs were 1‐butyl‐3‐methylimidazolium tetrafluoroborate, 1‐hexyl‐3‐methylimidazolium tetrafluoroborate and 1‐methyl‐3‐octylimidazolium tetrafluoroborate. The optical behaviour of heterocyclic aromatic amines in presence of ILs was studied and the silanol‐suppressing potency of ILs was evaluated for the two stationary phases studied. Several chromatographic parameters were evaluated in the presence or absence of ILs, or using triethylamine, the most common mobile‐phase additive. The best results were achieved using 1 mM 1‐butyl‐3‐methylimidazolium tetrafluoroborate as mobile‐phase additive and NovaPak^® column. In these conditions and with 18% of ACN in the mobile phase, analytical performance of the chromatographic methods using fluorescence and UV–Vis were evaluated, obtaining good precision in all cases (RSD lower than 6.6%) and low LOD (0.001–0.147 μg/mL with UV–Vis and 0.001–0.006 ng/mL with fluorescence detection).     

Analytical developments and applications of ionic liquids for environmental studies

Ionic liquids (ILs) are considered advanced solvents with interesting properties that have led to remarkable improvements in the performance of analytical methods and their practical application. Analytical chemistry has profited from the evolution of ILs in diverse contexts, ranging from their applications in microextractions to uses as matrices for mass spectrometric determinations. Their use in sample preparation has meant significant improvements in terms of miniaturization and analytical performance, and given place to new techniques based on liquid-liquid and solid-phase extractions; the latter greatly driven forward by the combination of ILs with nanomaterials. Furthermore, electrodes have been prepared by combining ILs with different modern materials, significantly improving the sensitivity and selectivity of electroanalytical methods. Moreover, the implementation of ILs as additives to mobile and stationary phases in separation techniques has been proved to improve liquid and gas chromatography, as well as capillary electrophoresis, in terms of the number of analytes that can be efficiently separated and of the useful life of columns, representing also a promising alternative to environmentally dangerous organic solvents. Additionally, their application as matrix modifiers and as ion-pairing additives has introduced their use in mass spectrometry. In this review, the design and implementation of innovative and highly efficient analytical methods based on ILs for the sensitive and selective determination of diverse analytes in environmental matrices is described. Critical issues that have arisen from their application and future challenges in electrochemical, separation and preconcentration techniques based on these solvents are also presented.     

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.     

Reversed-phase liquid chromatographic separation of antiretroviral drugs on a monolithic column using ionic liquids as mobile phase additives

The use of 3-methylimidazolium cation-based ionic liquids (ILs) was evaluated as mobile phase additives for separation of antiretroviral drugs on a monolithic column by RP-HPLC. Separation of eight commonly used antiretroviral drugs was achieved on a Chromolith Flash, RP-18e column (25 × 4.6 mm, porous material) using water (pH 4.0 adjusted with acetic acid)/methanol v/v as a mobile phase containing ILs in a gradient elution mode. The effects of concentrations of ILs on retention, resolution and peak shape were studied and a regression equation correlating the interactions between stationary phase and the ILs was established. The retention of all the drugs was decreased notably by using 1-butyl-3-methylimidazolium tetrafluoroborate, while 1-ethyl-3-methylimidazolium methylsulfate reduced gradient drift drastically when compared to triethylamine.     

Protein separation using a novel silica-based RPLC/IEC stationary phase modified with N-methylimidazolium ionic liquid

Ionic liquids (ILs) immobilized on silica as novel high performance liquid chromatography (HPLC) stationary phases have attracted considerable attention. However, it has not been applied to protein separation. In this paper, N-methylimidazolium IL-modified silica-based stationary phase (SilprMim) was prepared and investigated as a novel multi-interaction stationary phase charged positively for protein separation. The results indicate that all of the basic proteins tested cannot be absorbed on this novel stationary phase, whereas all of the acidic proteins tested can be retained, and the baseline separation of eight kinds of acidic protein standards can be achieved when performed in reversed phase/ ion-exchange chromatography (RPLC/IEC) mode. Compared with commonly used commercial octadecylated silica (ODS) column, the novel stationary phase can show selectivity and good resolution to acidic proteins, which has a promising application in the separation and analyses of acidic proteins from the complex samples in proteomics. In addition, the chromatographic behavior of proteins, the effect of the ligand structure and the retention mechanism on this stationary phase were also investigated.     

Separation Characteristics of Ionic Liquids Grafted Polymethylsiloxanes Stationary Phases for Capillary GC

Ionic liquids (ILs) grafted polymethylsiloxane (PMS) stationary phases (IL-PMS) for capillary gas chromatography (CGC) are described. The stationary phases were synthesized by grafting 1-vinyl-3-hexylimidazolium (VHIm) with either NTf ₂ ^? or PF₆ ^? anion to poly(methylhydrosiloxane) (PMS-VHIm-NTf₂, PMS-VHIm-PF₆) and coated statically onto fused-silica capillary columns. Separation characteristics of the stationary phases involving Abraham solvation parameters, separation ability and thermal stability were investigated. The obtained solvation parameters reveal that both IL-PMS stationary phases exhibited unique intermolecular interactions compared with either ILs or PMS due to the synergistic effect of ILs and PMS chemically combining together. The separation performance of the IL-PMS stationary phases was investigated by a Grob mixture and a complex mixture of 26 compounds of different types. The results show that the present stationary phases exhibit excellent resolution and selectivity for the analytes of interest with narrow and symmetric peak shapes. Thermal stability was also investigated by column bleed profiles with satisfactory results. The satisfactory chromatographic performance and thermal stability of the IL-PMS stationary phases suggest their great potential as a new type of CGC stationary phases.     

Reversed-phase liquid chromatography of alkaloids: Masking effects of inorganic salts

The addition of salts (investigated cations Na(+), NH(+)(4), K(+), KBu(+)(4) combined with the anions acetate, Br(-), SCN(-) and I(-)) can be used to eliminate peak tailing and to decrease retention of cationic species in the course of the separation of alkaloids from Chelidonium majus L. by reversed-phase chromatography on a ODS Hypersil column, using water-acetonitrile-methanol mixture as eluent. These findings are interpreted in terms of a silanol masking effect. The extended donor-acceptor concept is used to interpret the effectiveness of different salts in masking the active sites of the stationary phase.     

A Review of Ionic Liquids in Chromatographic and Electromigration Techniques

Although there have been numerous studies on the use of ionic liquids (IL) as solvents for synthesis and catalysis, there are many potential new fields for their application. The number of studies dealing with the use of ILs as additives to the mobile phase in LC and CZE and as a stationary phases in GC is constantly increasing. The main goal of the present paper is to gather together studies concerning the use of ILs in chromatographic techniques. The application of these substances as stationary phases, mobile phase additives and electroosmotic flow modifiers is discussed. Conversely, the application of separation methods in the analysis of ILs is also the subject of this review.     

Comparison of column properties in reversed-phase chromatography: monolithic, cholesterolic and mixed bonded stationary phases

A commercial Chromolith C18 column and two new stationary phases with mixed ligands bonded on the Kromasil silica gel support, SG-MIX and SG-Chol, were characterized using simple tests based on the retention of non-polar, basic and acidic compounds. Polar and methylene selectivity tests in acetonitrile-water and methanol-water mobile phases revealed lower hydrophobicities of the SG-MIX and SG-Chol columns in comparison to the Chromolith column. The columns were further characterized using new test criteria - gradient oligomer capacity and isomeric selectivity and peak symmetry of naphthalene di-sulphonic acids in aqueous mobile phases. The cholesterolic column shows greater gradient oligomer selectivity for the separation of oligoethylene glycol samples than the SG-MIX and the Chromolith columns. Increased retention and peak tailing, but decreased isomeric selectivity for naphthalene-di-sulphonic acids was observed with the SG-MIX column, because of interactions with various polar bonded groups.     

Retention characteristics of organic compounds on molten salt and ionic liquid-based gas chromatography stationary phases

The interest of using ionic liquids (ILs) as stationary phases in gas chromatography (GC) has increased in recent years. This is largely due to the fact that new classes of ILs are being developed that are capable of satisfying many of the requirements of GC stationary phases. This review highlights the major requirements of GC stationary phases and describes how molten salts/ILs can be designed to largely meet these needs. The retention characteristics of organic solutes will be discussed for ammonium, pyridinium, and phosphonium-based molten salts followed by imidazolium, pyridinium, pyrollidinium, and phosphonium-based IL stationary phases. The versatility of ILs allows for the development of stationary phases based on dicationic ILs, polymeric ILs, and IL mixtures. To aid in choosing the appropriate IL stationary phase for a particular separation, the reader is guided through the different types of stationary phases available to identify those capable of providing the desired separation selectivity of organic solutes while allowing for flexibility in ranges of temperature used throughout the separation.     

Selectivity of stationary phases based on pyridinium ionic liquids for capillary gas chromatography

A number of capillary columns with stationary liquid phases based on mono- and dication pyridinium ionic liquids (ILs) were prepared. Their polarity was evaluated using McReynolds system and the selectivity was estimated from intermolecular interactions. The parameters of intermolecular interactions were obtained from retention data using the (Abraham) model of the linear free energy relationship. The dependences of intermolecular interactions on the structure of the cation in the ILs under study were revealed. The results were compared with the data for the traditional phases (HP-5, ZB-WAX). Examples of separation of mixtures of oxygen-containing compounds on the phases under study are given.     

Comparison of Different Silica-Based Imidazolium Stationary Phases for LC in Separation of Alkaloids

Three stationary phases based on silica modified with imidazolium ionic liquids were prepared and characterized for their use in high-performance liquid chromatography. Multiple interactions of separation mechanism of the three stationary phases were studied by using caffeine, theophylline and theobromine. The comparison of differential retentions was established and the hydrophobic interaction of the alkylimidazolium groups showed stronger impact than other interactions in separation of alkaloids. The effects of temperatures and different concentrations of additives in eluent were also investigated. Furthermore, the study of retention mechanism was used to predict the order of elution of cytosine, thymine and guanine successfully.