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.     

Interpretation of interactions of halogenated hydrocarbons with modified silica adsorbent coated with 3-benzylketoimine group silane

The paper reports a new group of adsorbents obtained by modification of silica surface with silane containing 3-benzylketoimine groups with bonded Cu(II) and Ni(II) chlorides. The adsorbents obtained were subjected to a chromatographic study to establish the type of adsorbate-adsorbent interactions for aliphatic and aromatic halogenated hydrocarbons used as adsorbates. The following retention parameters were determined: retention factor (k), Kovats retention index (I), specific retention volume (V _g), and molecular retention index (ΔM _e). Values of the molecular retention index were subjected to complex statistical analysis providing qualitative relations between the adsorbates’ properties and spatial structure and their retention. It was observed that bonding of chlorides of Cu(II) and Ni(II) with the ketoimine groups resulted in an increase of the retention index and the molecular retention index indicating an increase of the adsorbate-adsorbent interaction in comparison with the reference column coated with silane including a 3-benzylketoimine group without transition metal chlorides. Along with the observed increase of the adsorbate-adsorbent interaction, larger difference between the retention indices was observed for both halogenated compounds. This indicates a positive influence of the introduced transition metals on the selectivity of the adsorbate-adsorbent interactions of the examined columns.     

Determination of hydrophobicity of organic bases on poly(butadiene)-coated alumina and on octadecylsilica columns

Summary The performance of two chromatographic (HPLC) methods recommended for hydrophobicity evaluation of structurally diverse (noncongeneric) solutes was compared. Azole derivative drugs possessing properties of weak organic bases were used as the test solutes. One of the methods, recommended by Minick and co-workers, consists on suppressing specific interactions with the stationary phase (ODS) by adding modifiers to the eluent of neutral pH. The other method, developed previously in our laboratory, yields retention data for nonionized bases due to using poly(butadiene)-coated alumina (PBCA) columns which can be operated under alkaline conditions. It has been demonstrated that in the case of basic solutes, hydrophobicity parameters obtained by the method employing PBCA columns are more reliable. The noncontrolled specific interactions of organic bases with the ODS phase at pH 7.0 remain effective in spite of special precautions undertaken.     

Chromatography Modeling Software Applicability for Non-Conventional Columns: a Case Study of Calixarene- and Resorcinarene-Bonded Stationary Phases

In this study a systematic evaluation of the applicability of DryLab for calixarene- and resorcinarene-bonded stationary phases and some other relatively new reversed-phase columns with the presence of conventional alkyl-bonded phases was carried out. Calixarene- and resorcinarene-bonded stationary phases belong to the reversed-phase materials. However, depending on the analytes, they show some additional interactions, since their steric, polar and ionic properties are different compared to those of conventional alkyl-bonded phases. Three different mixtures of model analytes, consisting of alkyl substituted benzene derivatives, 4-hydroxybenzoic acid esters and polycyclic aromatic hydrocarbons, were used to verify the accuracy of DryLab prediction of retention times and to compare the results of 20 different liquid chromatographic phases. The type and the content of the organic modifier as well as the temperature and the gradient time were systematically changed using same conditions for all stationary phases. The results showed that the prediction on the calixarene- and resorcinarene-bonded stationary phases as well as on other reversed-phase columns is highly accurate in both isocratic and gradient modes. The predictions and real experiments were highly correlated with an average absolute error (∆t _R) of 0.027 min (<2 s) and an average percent absolute error (%∆t _R) of 0.38 on the calixarene- and resorcinarene-bonded stationary phases, and ∆t _R of 0.04 min (<3 s); %∆t _R of 0.51 on the other reversed-phase columns in this study. As a result, DryLab could be applied with very accurate predictions in method development using calixarene- and resorcinarene-bonded stationary phases, which were used as an example for “new” stationary phase materials.

     

Chromatography Modeling Software Applicability for Non-Conventional Columns: a Case Study of Calixarene- and Resorcinarene-Bonded Stationary Phases

In this study a systematic evaluation of the applicability of DryLab for calixarene- and resorcinarene-bonded stationary phases and some other relatively new reversed-phase columns with the presence of conventional alkyl-bonded phases was carried out. Calixarene- and resorcinarene-bonded stationary phases belong to the reversed-phase materials. However, depending on the analytes, they show some additional interactions, since their steric, polar and ionic properties are different compared to those of conventional alkyl-bonded phases. Three different mixtures of model analytes, consisting of alkyl substituted benzene derivatives, 4-hydroxybenzoic acid esters and polycyclic aromatic hydrocarbons, were used to verify the accuracy of DryLab prediction of retention times and to compare the results of 20 different liquid chromatographic phases. The type and the content of the organic modifier as well as the temperature and the gradient time were systematically changed using same conditions for all stationary phases. The results showed that the prediction on the calixarene- and resorcinarene-bonded stationary phases as well as on other reversed-phase columns is highly accurate in both isocratic and gradient modes. The predictions and real experiments were highly correlated with an average absolute error (?t _R) of 0.027 min (<2 s) and an average percent absolute error (%?t _R) of 0.38 on the calixarene- and resorcinarene-bonded stationary phases, and ?t _R of 0.04 min (<3 s); %?t _R of 0.51 on the other reversed-phase columns in this study. As a result, DryLab could be applied with very accurate predictions in method development using calixarene- and resorcinarene-bonded stationary phases, which were used as an example for “new” stationary phase materials.     

Composite adsorbents based on inorganic salts for the rapid preconcentration of low-molecular polar organic compounds from humid air followed by gas chromatographic determination

The regularities of the retention of organic vapors on composite adsorbents based on inorganic salts are determined. It is found out that such adsorbents possess very high adsorption capacity relative to low-molecular polar organic compounds and can be used for their adsorption preconcentration from an air flow, followed by thermal desorption and gas-chromatographic determination. To reduce the detection limits of analytes in humid air, a two-column system of adsorption preconcentration is proposed, involving passage of an air sample through two columns connected in a series, one with a KF-based desiccant and another with a Mg(ClO₄)₂-based adsorbent.     

Determination of nitrite and nitrate in Venice lagoon water by ion interaction reversed-phase liquid chromatography

Ion interaction reversed-phase liquid chromatography with octylammonium orthophosphate as the interacting reagent and a reversed-phase C₁₈ column was applied to the identification and determination of nitrite and nitrate in Venice lagoon water. Interference by the high chloride concentration was systematically studied and the results obtained with different column packings were compared. With spectrophotometric detection at 230 nm, nitrite at 0.005 mg 1^?1 can be detected and determined even in the presence of 0.70 M chloride. The dependence of the retention time of nitrite on the chloride concentration was studied for two reversed-phase columns with different packings. Concentrations of 0.30 ± 0.05 mg 1^?1 of nitrite and 0.20 ± 0.05 mg 1^?1 of nitrate were found in Venice lagoon water.     

Analysis and critical comparison of the reversed-phase and ion-exchange contributions to retention on polybutadiene coated zirconia and octadecyl silane bonded silica phases

The two major modes of retention of basic compounds in reversed-phase liquid chromatography on both octadecyl silane bonded silica-based (ODS) and polybutadiene coated zirconia (PBD-ZrO2) materials are hydrophobic and ion-exchange (Coulombic) interactions. Although the influence of reversed-phase and Coulombic interactions on the chromatography of organic cations is qualitatively well recognized, the quantitative relationship between hydrophobic and ion-exchange interactions remains unclear. In this work, the retention mechanisms on both of the above types of phases were probed by studying the retention of a homologous series of p-alkylbenzylamines as a function of the ammonium concentration in the eluent. The various columns tested were studied in terms of plots of retention factor vs. the inverse of the displacingcation concentration. The analysis of such plots as well as plots of log k' vs. number of methylene groups in the solutes and plots of log k' vs. log[NH4+] clearly shows that at least two types of sites--a pure reversed-phase site and a "hydrophobically-assisted ion-exchange site" similar to the type of site described by Neue [J. Chromatogr. A 925 (2001) 49] are needed to explain the observations. In addition, we have found a quantitative measure of the relative amount of reversed-phase and ion-exchange interaction on a given solute has on a given stationary phase which allows unambiguous classification of columns. It is now clear that ion-exchange contributions to retention on PBD-ZrO2, sometimes exceeding 90%, are even more important than previously thought and relative to hydrophobic interaction much more significant on PBD-ZrO2 than on ODS type-B silicas.     

Ion-exchange properties of silica gel with covalently bonded histidine

The ion-exchange and acid-base properties of Silasorb 600 (10 μm) with immobilized histidine (His-SiO₂) were investigated. The acid-base properties of the sorbent were estimated from the dependence sof the retention of organic acids and bases on the eluent pH. The effect of various organic modifiers (benzoic, citric, oxalic, and sulfuric acids) and the eluent pH on the retention of inorganic ions was studied. It was shown that His-SiO₂ exhibits anion-exchange properties in acidic and weakly acidic media at pH < 5.7. Eight anions were separated under optimal conditions on a 150 × 4.6-mm column in 21 min using a 5 mM oxialic acid solution as an eluent.     

Retention of some glucosinolates in anion exchange chromatography. Part I: Qualitative study on a silica-trialkylammonium exchanger

Summary The retention and separation of glucosinolates, as organic anions, were studied on a silica-based strong anion exchanger under isocratic elution conditions. All glucosinolates carry the same functional ionic group (-OSO ₃ ^– ), however they do not have the same retention in anion exchange chromatography. The plots of capacity factors of organic anions versus the reciprocal of eluent ion concentration show good linearity. From the slope and y-intercept data the major retention mechanisms are interpreted as ion exchange and reversed-phase interactions. The effects of nature and concentration of the eluent ion and the influence of organic modifier addition to the aqueous buffered mobile phase are also investigated. Direct and indirect UV detection were used.Our results open the way for the development of new systems for intact glucosinolate analysis which are easier to use than the present ion-pairing chromatographic method.     

Factors Affecting the Interpretation of Selectivity on Synergi Reversed-Phase Columns

The solvation parameter model is used to establish the contribution of cohesion, dipole-type, and hydrogen-bonding interactions to the retention mechanism on Synergi Hydro-RP, Fusion-RP, and Polar-RP reversed-phase columns with methanol–water mobile phases containing from 10–70% (v/v) methanol. Large changes in relative retention on the compared columns can result from steric resistance, differences in the phase ratios, and from dewetting at low methanol compositions while changes in intermolecular interactions are responsible for smaller changes at a fixed mobile phase composition. For Synergi Hydro-RP and Polar-RP changing methanol for acetonirile is more powerful for affecting changes in retention order than changing the stationary phase. The three Synergi columns show useful selectivity differences for method development when compared with 13 other modern reversed-phase columns representing a selection of different stationary phase chemistries. The results from this study indicate the limitations of classifying reversed-phase columns by the retention of prototypical compounds to define specific retention mechanisms.     

Contributions to reversed-phase column selectivity. II. Cation exchange

The contribution of cation exchange to solute retention for type-B alkylsilica columns (made from high-purity silica) has been examined in terms of the hydrophobic-subtraction (H-S) model of reversed-phase column selectivity. The relative importance of cation exchange in the separation of ionized bases by reversed-phase chromatography (RPC) varies with (a) column acidity (values of the column cation-exchange capacity C), (b) mobile-phase pH and buffer concentration, and (c) the nature of the buffer cation. The effects of each of these separation variables on cation retention were examined. The contribution of cation exchange (and other ionic interactions) to solute retention is represented in the H-S model by properties of the solute (κ') and column (C), respectively. Values of κ' for 87 solutes have been examined as a function of solute molecular structure, and values of C for 167 type-B alkylsilica columns have been related to various column properties: ligand length (e.g., C(8) vs. C(18)) and concentration (μmol/m(2)), pore diameter (nm), and end-capping. These results contribute to a more detailed picture of the retention of cationic solutes in RPC as a function of separation conditions. While previous work suggests that the ionization of type-B alkylsilica columns is generally negligible with mobile-phase pH<7 (as a result of which cation exchange then becomes insignificant), the present study provides evidence for cation exchange (and presumably silanol ionization) at a pH as low as 3 for most columns.     

Separation of alicyclic and aromatic hydrocarbons on a PLOT column coated with 3-benzylketoiminepropyl group

An interpretation of specific electron-donor-acceptor interactions between the adsorbent coating the walls of a capillary column and adsorbates from the groups of alicyclic and aromatic hydrocarbons is proposed. The adsorbent was based on silica the surface of which was modified with silane containing 3-benzylketoimine groups to improve its adsorption properties. The columns studied had walls coated with 3-benzylketoimine groups, and Cu(II) chloride complexes or with Ni(II) chloride complexes. The adsorbate-adsorbent interactions were interpreted on the basis of the Kovats retention index, specific retention volume, molecular retention index, and ??M _e values. The influence of particular elements of spatial structure and the positions of double bonds in the adsorbate molecule was evaluated on the modelling calculations based on the quantitative structure-retention relationships. The introduction of transition metal ions in the form of complexes into the adsorbents studied increased the strength of the interactions between the adsorption layer of the capillary column and the adsorbate molecules. The increased strength of the interactions was accompanied by increased selectivity of the columns with regard to a group of alicyclic and aromatic hydrocarbons. The analytical performance of the columns thus obtained was compared with that of a commercial column coated with the 100 % dimethyl polysiloxane phase.     

A Study of the Retention Behaviour of Various Organometallic Complexes in HPLC Phase Systems

Abstract

The retention behaviour of various organometallic complexes in HPLC has been investigated. Three polar adsorbents (silica, a CN-bonded and a diol-bonded stationary phase) and a nonpolar adsorbent (C₁₈-bonded stationary phase) were used in this study for normal phase LC and reversed-phase LC respectively. The retention is influenced by the type of metal atom and the type of ligand in both types of LC. The study includes complexes of iron, ruthenium, cobalt, manganese, molybdenum and nickel with di-aza-butadiene and tetra-azadiene ligands. It was found that some ruthenium complexes are not eluted from bare and chemically modified silica.     

Molecular Theory of Liquid Adsorption Chromatography

Abstract

A statistical-mechanical theory, based on a lattice model, has been developed to address the molecular mechanism of retention and selectivity in both normal-phase and reversedphase liquid adsorption chromatography. The model is a natural “competitive-equilibrium” one, where possible contributions from solvent-solvent and solute-solvent interactions, and, hence, from solution nonideality, are not neglected. Homogeneous and heterogeneous adsorbent surfaces, single-solvent and binary mixed-solvent mobile phases, and solute molecules of different size and shape are treated. Practical applications of the theory are presented to demonstrate its utility and significance.

For homogeneous adsorbents and neat solvents, the molecular energetics of retention and selectivity are examined, with special emphasis on the effects of solute size and shape, and, relatedly, the modes of solute adsorption. Separations of geometrical isomers and homologous series in real and simulated chromatographic processes are investigated, confirming predictions of the theory and the important role of solvent-solvent and solute-solvent interactions in reversed-phase systems. The implications of a more general retention equation for microscopically heterogeneous adsorbents are discussed. The dependence of capacity ratio on mobile-phase composition for binary solvents is analyzed in some detail. An often important contribution arising from solution nonideality is predicted theoretically. This is shown to be consistent with experimental results on normal-phase and reversed-phase systems.     

Effect of stationary and mobile phases on hydrophobic interaction chromatography of proteins and peptides

A number of different stationary phases designed for hydrophobic interaction chromatography have been examined to assess their efficiency and resolving capability with respect to protein and peptide mixtures. A packing with an ether-bonded phase was substantially less hydrophobic than those with propyl- or phenyl-bonded surface chemistry. While the overall efficiencies of most columns were broadly similar with respect to most proteins, some proteins did chromatograph with enhanced efficiency on specific packings. The elution order of individual proteins was, with one or two exceptions, similar for all columns tested using comparable mobile phases. It differed, however, substantially from orders obtained with conventional reversed-phase alkyl-bonded phases and from the elution orders obtained when the hydrophobic packings were used in a reversed-phase mode, i.e. with an organic modifier gradient. Varying the salt used in the mobile phase and its pH under hydrophobic interaction conditions (high ionic strength) changed overall retentivities and also altered specific retention orders, thus offering possibilities of selective resolution of some mixtures.     

Characterization of different RP-HPLC columns by a gradient elution technique

Summary The literature contains no unified testing procedure for evaluation and characterization of reversed-phase (RP) columns of different pore size (surface area) and containing different types of ligand. In our laboratory a testing procedure has been developed using gradient elution under standardized conditions independent of column dimensions and applicable to both narrow-pore and wide-pore columns. Six wide-pore and four narrow-pore columns were investigated and compared. The test solutes were selected to cover a wide range of chemical properties. The evaluation and characterization of the columns was performed on the basis ofk _app retention factors. Principal components analysis (PCA) was performed to reveal similarities and differences among the columns and test substances. The factors obtained characterize the columns according to the extent of various interactions such as hydrophobicity, HB (hydrogen bond)-donor acidity and HB-acceptor basicity. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Comparison of a C30 Bonded Silica Column and Columns with Shorter Bonded Ligands in Reversed-Phase LC

The Carotenoid S is a new C₃₀ bonded silica stationary phase, intended for reversed-phase chromatographic applications, which is more hydrophobic and consequently shows stronger retention in comparison to conventionally used C₁₈ stationary phases. We compared the non-polar selectivities of the columns for homologous alkylbenzenes in acetonitrile—water and methanol–water mobile phases and polar reversed-phase selectivities employing the interaction indices and the Linear Free Energy Relationship models. Further, we investigated possibilities of separations of structurally closely related compounds in the groups of phenolic acids, flavones, phthalic acids and related compounds and of acylglycerols on the new C₃₀ column and with different types of columns for reversed-phase chromatography, including shorter alkyl C₄, C₈, C₁₈ and phenyl bonded stationary phases. The C₃₀ column has in some aspects properties similar to the non-endcapped Nova-Pak column for separation of some acylglycerols with equal equivalent carbon numbers, but enables separations of longer chain triacylglycerols in a single gradient run.