Insights into the retention mechanism of neutral organic compounds on polar chemically bonded stationary phases in reversed-phase liquid chromatography

The solvation parameter model is used to characterize the retention properties of a 3-aminopropylsiloxane-bonded (Alltima amino), three 3-cyanopropylsiloxane-bonded (Ultrasphere CN, Ultremex-CN and Zorbax SB-CN), a spacer bonded propanediol (LiChrospher DIOL) and a multifunctional macrocyclic glycopeptide (Chirobiotic T) silica-based stationary phases with mobile phases containing 10 and 20% (v/v) methanol-water. The low retention on the polar chemically bonded stationary phases compared with alkylsiloxane-bonded silica stationary phases arises from the higher cohesion of the polar chemically bonded phases and an unfavorable phase ratio. The solvated polar chemically bonded stationary phases are considerably more hydrogen-bond acidic and dipolar/polarizable than solvated alkylsiloxane-bonded silica stationary phases. Selectivity differences are not as great among the polar chemically bonded stationary phases as they are between the polar chemically bonded phases and alkylsiloxane-bonded silica stationary phases.     

Separation of polycyclic aromatic hydrocarbons with a C60 bonded silica phase in microcolumn liquid chromatography

A chemically bonded C₆₀ silica phase was synthesized as a stationary phase for liquid chromatography (LC) and its retention behavior evaluated for various polycyclic aromatic hydrocarbons (PAHs) using microcolumn LC. The results indicate that the C₆₀ bonded phase offers selectivity different from that of octadecylsilica (ODS) bonded phases in the separation of isomeric PAHs. With the C₆₀ phase, PAH molecules having a partial structure similar to that of the C₆₀ molecule, e.g. triphenylene and perylene, were retained longer than with ordinary ODS stationary phases. The results also show that good correlation exists between the retention data with this C₆₀ bonded phase and with C₆₀ itself as the stationary phase.     

Retention parameters of aromatic hydrocarbons with mono-substituted polar groups in binary RP-HPLC systems

Summary Capacity factor (k′) values of aromatic hydrocarbons with mono-substituted polar-groups are correlated for reversed-phase systems involving stationary phases with C₁₈ or C₄ ligands chemically bonded to silica and a binary aqueous eluent containing modifiers: methanol, acetonitrile, tetrahydrofuran, isopropanol, dioxane or dimethoxyethane. The relative retention variations of the solutes are interpreted with special consideration of their interactions with non-polar stationary phases and the molecular structure of the modifiers and solutes. Rules for retention and selectivity optimisation in RP-HPLC systems are given.     

Cyclam complexes of Cu(II) and Co(II) as stationary phases for gas chromatography

New macrocyclic stationary chemically bonded phases were synthesized and tested in gas chromatography conditions. The complexes of 1,4,8,11-tetraazacyclotetradecane with Cu(II) and Co(II) were bonded to the silica support through the (3-chloropropyl)triethoxysilane reactant. The packings obtained were analyzed by diffuse-reflectance ultraviolet–visible spectroscopy (DRUV–Vis), differential thermal gravimetry (DTG), porosimetry, and elementary analysis. Preliminary study of the novel silica gas chromatography (GC) stationary phases containing cyclam complexes was carried out using packed 1/8 in. i.d. columns. The study was conducted on: cyclic, linear and branched olefins, aromatic hydrocarbons and ethers. Characterization of interactions between the compounds mentioned and new stationary phases was based upon analysis of Kováts retention indices (I), difference between retention indices for two phases (ΔI), and molecular retention indices (ΔM_e). Results have shown that the new stationary phases interact sufficiently strongly with molecules of high electron density and can be applied in capillary gas chromatography for the analysis of light hydrocarbons.     

Evaluation and comparison of n‐alkyl chain and polar ligand bonded stationary phases for protein separation in reversed‐phase liquid chromatography

Protein retention is very sensitive to the change of solvent composition in reversed‐phase liquid chromatography for so called “on–off” mechanism, leading to difficulty in mobile phase optimization. In this study, a novel 3‐chloropropyl trichlorosilane ligand bonded column was prepared for protein separation. The differences in retention characteristics between the 3‐chloropropyl trichlorosilane ligand bonded column and n‐alkyl chain modified (C₂, C₄, C₈) stationary phases were elucidated by the retention equation . Retention parameters (a and c) of nine standard proteins with different molecular weights were calculated by using homemade software. Results showed that retention times of nine proteins were similar on four columns, but the 3‐chloropropyl trichlorosilane ligand bonded column obtained the lowest retention parameter values of larger proteins. It meant that their retention behavior affected by acetonitrile concentration would be different due to lower |c| values. More specifically, protein elution windows were broader, and retentions were less sensitive to the change of acetonitrile concentration on the 3‐chloropropyl trichlorosilane ligand bonded column than that on other columns. Meanwhile, the 3‐chloropropyl trichlorosilane ligand bonded column displayed distinctive selectivity for some proteins. Our results indicated that stationary phase with polar ligand provided potential solutions to the “on–off” problem and optimization in protein separation.     

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.

     

Survey and Trends in the Preparation of Chemically Bonded Silica Phases for Liquid Chromatographic Analysis

In order to increase chromatographic selectivity and to extend the analytical capability of reversed phase liquid chromatography (RP HPLC) many investigators have concentrated on the preparation of silica based column packings with chemically bonded phases (CBP). These phases have also been successfully used in sample preparation techniques, mainly in solid phase extraction (SPE). Although alkyl bonded phases (e.g., C₂, C₈, and C₁₈) are the most widely used packings in RP HPLC and SPE, various specific applications require CBPs with polar functional groups (e.g., -NH₂, -NO₂, -CN, and/or -OH). The solution of problems with separation of complicated chiral compounds was attempted by applying stationary phases with chiral selectors (e.g., cyclodextrins, Pirkle phases, crown ethers, etc.). On the other hand, packings with pseudo-membrane or liquid crystal properties have been utilized for the separation of various substances of natural origin. Porous silica is commonly used as a support in the preparation of CBPs. Its physico-chemical characteristics, such as: type and structure of siliceous matrix, porosity, type and concentration of silanol groups, as well as surface purity, strongly influence the density and structure of chemically bonded phases. Recognition of these properties is helpful in optimizing separation processes based on RP HPLC elution and/or extraction of substances with polar character.     

Study of RP HPLC Retention Behaviours in Analysis of Carotenoids

For determination of selected carotenoids, various types of columns for high-performance liquid chromatography (HPLC) with different properties have been used. The characteristics of the laboratory-used packing material containing monomeric alkyl-bonded phases (C₁₈, C₃₀) and phenyl as well as phenyl-hexyl stationary phases were studied. The retention data of the examined compounds were used to determine the hydrophobicity and silanol activity of stationary phases applied in the study. The presence of the polar and carboxyl groups in the structure of the bonded ligand strongly influences the polarity of the stationary phase. Columns were compared according to methylene selectivity using a series of benzene homologues. The measurements were done using a methanol–water mobile phase. Knowledge of the properties of the applied stationary phase provided the possibility to predict the RP HPLC retention behaviours in analysis of carotenoids including lutein, lycopene and β-carotene. The composition of the mobile phase, the addition of triethylamine and the type of stationary phase had been taken into account in designing the method of carotenoid identification. Also a monolithic column characterised by low hydrodynamic resistance, high porosity and high permeability was applied. The presented results show that the coverage density of the bonded ligands on silica gel packings and length of the linkage strongly influence the carotenoid retention behaviours. In our study, the highest retention parameters for lutein, lycopene and β-carotene were observed for C₃₀ and C₁₈ stationary phase. This effect corresponds with pore size of column packing greater than 100 Å and carbon content higher than 11 %.     

Use of Chemically Bonded β‐Cyclodextrin Silica Stationary Phase for Liquid Chromatographic Separation of Structural Isomers

The retention behavior of five disubstituted benzene derivatives and two naphthalene derivatives is examined by using a chemically bonded β‐cyclodextrin silica stationary phase with the moiety containing the s‐triazine. The chromatographic results of five disubstituted benzene derivatives and two naphthalene derivatives show that effective separation is achieved on this stationary phase by high‐performance liquid chromatography. The results of the present investigation indicate that the formation of inclusion complexes plays a dominant role in the separation mechanism. However, the selectivity can be significantly enhanced by the n‐n interactions between the s‐triazine ring of the chemically bonded β‐cyclodextrin silica stationary phase and the aromatic ring of solutes. For example, the effective separation of the o‐, m‐, and p‐toluidine isomers on this stationary phase with the moiety containing the s‐triazine ring was better than on that of some β‐cyclodextrin bonded stationary phases without the moiety containing s‐triazine ring.     

Separation of Dietary Folates by Gradient Reversed-Phase HPLC: Comparison of Alternative and Conventional Silica-Based Stationary Phases

A study of ten silica-based stationary phases and gradient elution conditions to separate dietary folates by reversed-phase HPLC was performed. Alkyl-bonded stationary phases (both conventional and alternative) were found to be the most promising for the separation of different folate monoglutamates in terms of selectivity and peak shape. These phases were better than phenyl-bonded phases which lacked selectivity when separating 10-formyl-folic acid and 5-formyl-tetrahydrofolate. Polar-bonded (cyano) stationary phase showed similar retention characteristics as the conventional alkyl-bonded phases, but ranked below those in terms of peak shape. Overall, alternative stationary phases exhibited slightly higher retention of late-eluted folates and greater retention variability for early-eluting tetrahydrofolate and 5-methyl-tetrahydrofolate. Best selectivity was achieved on alternative polar endcapped Aquasil C₁₈ followed by conventional Synergy MAX C₁₂ and Genesis C₁₈ stationary 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.     

Capillary complexation gas chromatography in analysis of cyclic and aromatic hydrocarbons

Summary Results of studies on stationary phases containing copper(II), nickel(II) and cobalt(II) chloride chemically bonded via cyano—or thiol groups are presented. The retention parameters—i.e. retention factor (k), retention index (I) and molecular retention index (ΔMe) and specific retention volume (Vg)-enabled the characteristics of specific interaction between aromatic and cyclic hydrocarbons and metal complexes chemically bonded to stationary phases to be determined. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

Comparison of the Separation Characteristics of the Organic–Inorganic Hybrid Stationary Phases XBridge C8 and Phenyl and XTerra Phenyl in RP-LC

Differences in the system constants of the solvation parameter model and retention factor correlation plots for varied solutes are used to study the retention mechanism on XBridge C₈, XBridge Phenyl and XTerra Phenyl stationary phases with acetonitrile–water and methanol–water mobile phases containing from 10 to 70% (v/v) organic solvent. These stationary phases are compared with XBridge C₁₈ and XBridge Shield RP₁₈ characterized in an earlier report using the same protocol. The XBridge stationary phases are all quite similar in their retention properties with larger difference in absolute retention explained by differences in cohesion and the phase ratio, mainly, and smaller changes in relative retention (selectivity) by the differences in individual system constants and their variation with mobile phase type and composition. None of the XBridge stationary phases are selectivity equivalent but XBridge C₁₈ and XBridge Shield RP₁₈ have similar separation properties, likewise so do XBridge C₈ and XBridge Phenyl, while the differences between the two groups of two stationary phases is greater than the difference within either group. The limited range of changes in selectivity is demonstrated by the high coefficient of determination (>0.98) for plots of the retention factors for varied compounds on the different XBridge phases with the same mobile phase composition.     

Factor analysis and experiment design in high-performance liquid chromatography. IX. Silica vs. chemically bonded phases in normal phase-HPLC

Summary The retention of a group of 38 E-s-cis and Z-s-cis chalcones on silica vs. nine polar chemically bonded phases is discussed. It was established that the relatively greatest similarity to silica is observed with the NH₂, DIOL and CN phases, whereas the chargetransfer type phases and the NO₂ one offer a different separation selectivity.     

Liquid chromatography of carbon clusters

Summary A comparison of three binary mobile phases in LC separation of C₆₀ and C₇₀ fullerences on chemically bonded 2,4-dinitroanilinopropyl (DNAP) stationary phase was carried out, n-Hexane-benzene has been found to be the best mobile phase for efficient separation of the all-carbon molecules permitting high loads in preparative LC.     

Preparation of a polyglycol-C8 bonded phase and its characteristics

Summary A new bonded phase containing both non-polar C₈ and polar polyglycol chains was synthesized in two steps. Silica was first silanized with octyltrimethoxysilane and then the intermediate product was reacted with tetraethylene glycol to introduce the polar group. The bonded phase exhibits a selectivity different from that of C₈ or C₁₈ reversed phases with a mixed-mode retention mechanism. The silanol effect on the polyglycol-C₈ bonded phase was examined with the test mixture proposed by Engelhardt and co-workers. The experimental results indicate that the new bonded phase displays less silanol effect. The observed tailing of some ionizable species resulted mainly from their partial dissociation at the pH values of eluents close to thepK _a values of the solutes. Some potential applications of the bonded phase are also given.     

Retention of cyanoalkanes and cyanoalkylbenzenes by bonded stationary phases in supercritical fluid chromatography

Summary The retention of cyanoalkanes and cyano-alkylbenzenes during SFC was investigated on alkyl and cyanoalkyl-bonded stationary phases and compared to alkane retention. The particular behaviour of short chain homologues was attributed to a silanophilic interaction with surface-OH groups on the silica and inhibition of specific interactions between solutes and C₁₀CN bonded groups.     

Normal- and Reversed-Phase Behaviour of 16,17-Secoestrone Derivatives on Cyanopropyl-Bonded Silica Gel

The retention behaviour of a number of 16,17-secoestrone derivatives has been studied by LC and HPTLC on a polar cyanopropyl-bonded stationary phase using non-aqueous and aqueous-organic mobile phases. The retention behaviour has been discussed in terms of nature of the solute, eluent and stationary phase. The correlation between retention constants of 16,17-secoestrone derivatives obtained from reversed-phases and commercially available ACD log P software (Advanced Chemistry, Toronto, Canada) has also been examined.     

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.