Effect of stationary phase hydrophobicity and mobile phase composition on the separation of carboxylic acids in ion chromatography

In a previous work, we studied the retention behavior of monovalent and divalent carboxylic acids on a highly cross-linked polystryene-divinylbenzene anion-exchange column (IonPac AS4A-SC) using a carbonate-based buffer, and a retention model was applied to the chromatographic data obtained. In this work we characterized the retention of carboxylates (formic, acetic, propionic, lactic, pyruvic, oxalic, malonic, succinic, fumaric, maleic, tartaric, glutaric, adipic, malic, mucic, trans-beta-hydromuconic, trans,trans-muconic acids) on a column with higher hydrophilicity (IonPac AS11) according to analyte and stationary phase properties, using previously investigated eluent compositions and comparing the retention data obtained. Moreover, the effect of organic modifiers (CH3OH and CH3CN) in the eluent on the retention factors was also evaluated. The chromatographic data obtained on the IonPac AS11 column were fitted by the retention model and allowed one to obtain and to compare ion-specific selectivity constants (parameters of the model) with the ones obtained with the previous column.     

High-performance liquid chromatography utilization of ionic liquids as mobile phase additives for separation and determination of the isomers of amino benzoic acids

For the isomers of amino benzoic acid, including o-, m-, p-amino benzoic acid, the beneficial effects of using the ionic liquid, 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIm][BF₄]), as mobile phase additives on retention behavior and separation were investigated. Chromatographic separation of the o-, m-, p-amino benzoic acid was performed on a reversed-phase C18 column by ultraviolet detection at 245 nm. The effects of several chromatographic parameters, concentrations and pH values of [BMIm][BF₄] solutions, methanol concentration and length of alkyl chain on different ionic liquids, on the separation and determination of the isomers were evaluated. The optimized chromatographic conditions were established using an aqueous 0.5 μmol/L [BMIm][BF₄] solution (pH 3.0)/methanol (40:60, v/v) as mobile phase without need of gradient elution, with separation of three amino benzoic acids achieved within four min. The calibration curve showed good linearity over the tested range of 2 mg/L to 120 mg/L for the three isomers with a correlation coefficients of 0.9999. The recoveries of the three amino benzoic acids of spiked components were between 99.8% and 100%. The method has been successfully applied to the determination of p-amino benzoic acid in the pharmaceutical, Bromine Mitag Procaine Injection.     

The effect of buffer concentration and cation type in the mobile phase on retention of amino acids and dipeptides in hydrophilic interaction liquid chromatography

The current work is focused on exploring the effect of buffer cation type and its concentration on retention of amino acids, dipeptides and their blocked analogues on two stationary phases, i.e., bare silica and amide-based in hydrophilic interaction liquid chromatography. Five different buffers of pH 4.0 composed of Tris/acetic acid, triethylamine/acetic acid, ammonium/acetic acid, Li⁺/acetic acid and Ba²⁺/acetic acid were used in various concentrations. Interestingly, an increase of the buffer concentration caused increasing, decreasing or stable retention of analytes, according to the cation type in the buffer. The buffers containing barium cations provided the highest retention of all the analytes in comparable mobile phases, i.e., buffers with the same ionic strength and pH on both columns. Moreover, using buffers with barium cation different selectivity for dipeptides was observed. The chromatographic systems with buffers consisting of triethylamine behaved differently compared to others.     

The effects of temperature and mobile phase on the retention of aliphatic carboxylic acids in hydrophilic interaction chromatography on zwitterionic stationary phases

The influence of the mobile phase and temperature, on the retention behavior of seven aliphatic acids (pyruvic, gluconic, 2‐oxoglutaric, tartaric, malic, oxalic, and citric acid) in hydrophilic interaction liquid chromatography on zwitterionic stationary phases with sulfobetaine and phosphorylcholine ligands is investigated. In agreement with the van't Hoff model, most acids show linear ln k versus 1/T plots. However, the retention of structurally symmetrical oxalic and tartaric dicarboxylic acids is almost independent of temperature, or slightly increases at rising temperature. The experimental parameters of the van't Hoff plots suggest positive entropic contributions to the retention of these symmetrical acids, possibly connected with changes in molecular symmetry on their adsorption. The type of the zwitterionic stationary phase and the mobile phase composition (the molar concentration of acetate buffer and the volume fraction of acetonitrile) affect the retention and the selectivity of the separation of the acids.     

The retention behaviour of amino acids in hydrophilic interaction liquid chromatography on zwitterionic stationary phases†

The retention behaviour of amino acids was studied in hydrophilic LC on zwitterionic stationary phases. Evaluation of the influences of acetonitrile/water content, ammonium acetate (NH₄Ac) concentration and mobile phase pH values was performed. Fourteen amino acids were tested and they were all retained to varying extents, with poorer retention in high water content eluents. The linear relationship between the logarithm of retention factor and log(water content) indicated that adsorption dominated or at least was partly involved in the separation mechanism. Electrostatic and hydrophilic interactions also contributed to the retention of these amino acids under different separation conditions with various mobile phase pH values and NH₄Ac concentrations. Thus, the overall retention mechanism could be explained as a combination of adsorption, electrostatic and hydrophilic interactions. The magnitude and contribution of each mechanism is dependent on the nature of the analyte and the separation conditions applied.     

The retention behaviour of conjugated bile acids in reversed phase high performance liquid chromatography.

The retention behaviour of conjugated bile acids has been studied in a reversed phase high performance liquid chromatographic (RP-HPLC) system by using the mixture of methanol and aqueous phosphate buffer as the mobile phase. The retentions of the conjugates in RP-HPLC have been found to be mainly controlled by the glycine and taurine groups. The selectivity between five different glycine and taurine conjugated bile acids is a constant in RP-HPLC. This selectivity has been used for peak identification in the practical separation of conjugated bile acids.     

Use of beta-cyclodextrin bonded phase with s-triazine moiety in the spacer for separation of aromatic carboxylic acid isomers by high-performance liquid chromatography

The separation and retention behavior of five aromatic carboxylic acid isomers was investigated by means of high-performance liquid chromatography (HPLC) using a β-cyclodextrin bonded phase with s-triazine ring in the spacer. The influence of mobile phase pH on the retention was examined. The presence of s-triazine moiety in the spacer enhances greatly the selectivity of the isomers of aromatic carboxylic acids. Baseline separations of the five aromatic carboxylic acid isomers were achieved. In particular, the isomers of toluic, aminobenzoic, nitrobenzoic and hydroxybenzoic acid were successfully and effectively separated. The chromatographic results indicate that, in addition to inclusion complexation, π-π interaction and hydrogen bonding interaction between the bonded phase and analytes play significant roles in the retention of these acid isomers. Different elution orders were observed for these acidic solutes with different substituents. Possible retention mechanisms are discussed.     

Influence of temperature and mobile phase composition on retention properties of oligomeric bonded phases in reversed-phase liquid chromatography (RPLC)

Summary The effect of temperature and mobile phase composition (methanol-water) on the retention behaviour of an oligomeric series of n-octylsilyl bonded phases in reversed-phase liquid chromatography has been investigated. Plots of lnk against 1/T (van't Hoff plot) and the enthalpy of transfer (ΔH^o) yields linear relationships under the conditions studied. The ΔH^o values of the aromatic hydrocarbons and n-alkyl benzoates are higher than those of the polar compounds due to their higher level of interaction with the stationary phase. A linear plot of ΔH^o vs. ΔS^o suggest that the retention process, which is essentially controlled by non-specific (dispersive) interactions between the solutes and the bonded ligands, is identical for all cases evaluated. The existence of similar retention mechanisms is confirmed by the constant value of the enthalpy-entropy compensation temperature of the columns for a given class of componds. As expected, decreasing the methanol content (% v/v) of the mobile phase results in increased eluite retention times. The methylene and phenyl selectivities are found to be independent of the carbon content of the stationary phases and varied only with the eluent composition. In addition to their high stability under aggressive mobile phase conditions as previously reported, the results of this study generally showed that the solute retention process on oligomeric phases are similar to those exhibited by the conventional reversed phases.     

Effect of the alkyl chain length of the bonded stationary phase on solute retention in reversed-phase high-performance liquid chromatography

Summary A comparison of the chromatographic retention characteristics of nonpolar bonded stationary phases was investigated. The results show that the interactions between the solutes and the stationary phases having C₂, C₈ and C₁₈ alkyl groups are almost similar in the range of the mobile phase investigated. This interaction is considered as the solvophobic effect.     

Influence of inorganic mobile phase additives on the retention and separation efficiency of selected amino acids in thin-layer chromatography on cellulose layers

Selected amino acid standards are investigated on cellulose layers using organic-aqueous eluent systems modified with neutral and chaotropic salts: chlorides, iodides, nitrates, thiocyanates, perchlorates, and hexafluorophosphates at low concentrations ranging from 10 up to 80mM in whole mobile phase. The effect of salts used as the mobile phase modifier is analyzed by the comparison of densitograms, peak symmetry coefficient, and theoretical plate number. The efficiency of chromatographic systems modified with inorganic salts additives depends primarily on the kind of salt and organic solvent in the mobile phase. The best efficiency is obtained through the addition of ammonium thiocyanate to the mobile phase containing acetonitrile as an organic modifier.     

Theory of retention in reversed phase liquid chromatography with ternary mobile phases

The partition model of retention is developed for reversed phase liquid chromatography with multicomponent mobile phases. Simple equations for the retention and selectivity in ternary mobile phases are derived. For the systems in which the ratio of volume fractions of organic modifiers remains fixed, new linear dependences for retention factor and selectivity are proposed. These equations are successfully used to describe experimental data found in the literature. An influence of the nature of organic solvents and proportion in which they are mixed on retention and selectivity is discussed.     

Effect of mobile phase composition on the retention of selected alkaloids in reversed-phase liquid chromatography with chaotropic salts

Sodium hexafluorophosphate, perchlorate and trifluoroacetate were applied as ion-ion interaction reagents in reversed-phase liquid chromatography. The separation of chosen alkaloids was performed by changing the kind of the organic modifier (methanol, acetonitrile, tetrahydrofuran), concentration of the ion-ion-interaction reagents and the concentration of phosphate buffer at constant pH (2.7) in the mobile phase. Obtained results were analyzed in connection to a dynamic ion-exchange model of retention and ion-ion interaction effects. The perturbation method was applied to test proposed retention theories. The formation of ion-complexes controlling the retention in chaotropic systems was confirmed. On the basis of the relationships of capacity factors (k) versus salt concentrations derived experimentally, absolute increases in capacity factors, the desolvation parameters and the limiting retention factors were calculated and compared for all the investigated compounds in eluent systems studied. The selectivity of the proposed mobile phases was compared on the basis of the separation of alkaloid mixture.     

Studies on the retention behaviour of some thiazolylazo derivatives in reversed phase liquid chromatography

The retention behaviour of thiazolylazo derivatives, 4-(2-thiazolylazo) resorcinol (TAR), 4-(2-thiazolylazo)-orcinol (TAO), 2-(2-thiazolylazo)-4-methylphenol (TAC) and 1-(2-thiazolylazo)-2-naphtol (TAN) was studied by reversed phase liquid chromatography. The optimum conditions for the separation of four thiazolylazo derivatives were examined with respect to column, flow rate, mobile phase composition and pH of mobile phase. These derivatives were separated simultaneously on Symmetry C₈ column using composition of acetonitrile/water (60/40, v/v) as mobile phase. The capacity factor (k′) has been decreased at higher pH than pK_a of solute which may due to the increasing concentration of the ionized species as increase the pH of mobile phase. The dependence of log k′ on the volume faction of water in the binary mobile phase and k′ on the liquid–liquid extraction distribution ratio (D_c) in acetonitrile–water (60/40, v/v)/n-octane extraction system for thiazolylazo derivatives were obtained good linear relationship. The results showed that the retention behaviour of these derivatives was mainly affected by the hydrophobic interaction between thiazolylazo as solute and mobile phase.     

Effect of mobile phase pH on the retention of nucleotides on different stationary phases for high-performance liquid chromatography

The main aim of the present investigation was to study the retention and separation of eight nucleotides with the use of seven stationary phases in RP HPLC mode. Two octadecyl columns were used; however, aminopropyl, alkylamide, cholesterol, alkyl-phosphate, and phenyl were also studied. Special attention was paid to the mobile phase buffer pH, since it appears that this factor is very influential in the case of chromatographic separation of nucleotides. The retention of nucleotides was greater for mobile phase pH?4.0 in comparison with pH?7.0 (except for octadecyl and phenyl packing). This is a consequence of protonization of polar groups present on the stationary phase surface. It was proved that aminopropyl, alkyl phosphate, alkylamide packing materials are not suitable for the resolution of nucleotides. On the other hand, cholesterol and phenyl stationary phases are alternatives for conventional octadecyl phases. Application of cholesterol packing allows separation of small, polar nucleotides, which is impossible to achieve in the case of octadecyl phase. Moreover, a phenyl support allows separation of nucleotides in a shorter time in comparison with octadecyl packing.     

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.     

Effects of ionic liquid as additive and the pH of the mobile phase on the retention factors of amino benzoic acids in RP-HPLC

As an organic salt, ionic liquids are widely used as new solvent media. In this paper, three positional isomers, such as o-amino benzoic acid, m-amino benzoic acid, and p-amino benzoic acid are separated with four different ionic liquids as additives to the mobile phase using reversed-phase (RP) high-performance liquid chromatography (HPLC). Amino benzoic acids are biologically active substances; the p-isomer is present in a group of water-soluble vitamins and is widely known as a sunscreen agent. The ionic liquids used are 1-butyl-3-methylimidazolium tetrafluoroborate, 1-ethyl-3-methylimidazolium tetrafluoroborate, 1-ethyl-3-methylimidazolium methylsulfate, and 1-octyl-3-methylimidazolium methylsulfate. The effects of the length of the alkyl group on the imidazolium ring and its counterion, the concentrations of the ionic liquid, and the effect of the pH of the mobile phase on the retention factor of the amino benzoic acid isomers are studied. Separation with the ionic liquid in the eluent was better than the separation without the ionic liquid. The pH mainly affected the retention and elution order of the solutes in RP-HPLC.     

Effect of water on the retention on diol and amide columns in hydrophilic interaction liquid chromatography

The amount of water adsorbed on polar columns plays important role in hydrophilic interaction liquid chromatography. It may strongly differ for the individual types of polar columns used in this separation mode. We measured adsorption isotherms of water on an amide and three diol‐bonded stationary phases that differ in the chemistry of the bonded ligands and properties of the silica gel support. We studied the effects of the adsorbed water on the retention of aromatic carboxylic acids, flavonoids, benzoic acid derivatives, nucleic bases, and nucleosides in aqueous‐acetonitrile mobile phases over the full composition range. The graphs of the retention factors versus the volume fraction of water in mobile phase show “U‐profile” characteristic of a dual hydrophilic interaction–reversed phase retention mechanism. The minimum on the graph that marks the changing retention mechanism depends on the amount of adsorbed water. The linear solvation energy relationship model suggests that the retention in the hydrophilic interaction liquid chromatography mode is controlled mainly by proton–donor interactions in the stationary phase, depending on the column type. Finally, the accuracy of hydrophilic interaction liquid chromatography gradient prediction improves for columns that show a high water adsorption.     

Effects of stationary-phase polarity on retention in reversed bonded phase HPLC columns

Summary Variations in retention and selectivity have been studied in cyano, phenyl and octyl reversed bonded phase HPLC columns. The retention of toluene, phenol, aniline and nitrobenzene in these columns has been measured using binary mixtures of water and methanol, acetonitrile or tetrahydrofuran mobile phases in order to determine the relative contributions of proton donor-proton acceptor and dipole-dipole interactions in the retention process. Retention and selectivity in these columns was correlated with polar group selectivities of mobile phase organic modifiers and the polarity of the bonded stationary phases. In spite of the prominent role of bonded phase volume and residual silanols in the retention process, each column exhibited some unique selectivities when used with different organic modifiers.     

Effect of stationary phase polarity on the retention of ionic liquid cations in reversed phase liquid chromatography

Chromatographic analysis of ionic liquids on different types of packings offers interesting possibility to determine their retention mechanism. As a consequence, the major interactions between stationary phase ligands and analyzed chemical entities can be defined. The main aim of this work was to analyze cations of ionic liquids on chemically bonded stationary phases with specific structural properties. The attempt to predict the main interactions between positive ions of ionic liquids and stationary phase ligands was undertaken. For that purpose, butyl, octyl, octadecyl, phenyl, aryl, mixed, alkylamide, and cholesterolic packings were chosen and applied to the analysis of six most commonly used ionic liquids' cations. Obtained results indicate mainly dispersive and pi-pi type of interaction part in the retention mechanism of analyzed compounds.