Phenyl‐bonded stationary phases—The influence of polar functional groups on retention and selectivity in reversed‐phase liquid chromatography

The chromatographic properties of four phenyl‐bonded phases with different structures were studied. The columns used were packed with a stationary phase containing a phenyl ring attached to the silica surface using different types of linkage molecules. As a basic characteristic of the bonded phases, the hydrophobicity and silanol activity (polarity) were investigated. The presence of the polar amino and amide groups in the structure of the bonded ligand strongly influences the polarity of the bonded phase. Columns were compared according to methylene selectivity using a series of benzene homologues and according to their shape and size selectivity using polycyclic aromatic hydrocarbons. The measurements were done using methanol/water and acetonitrile/water mobile phases. The presented results show that the presence of polar functional groups in the ligand structure strongly influences the chromatographic properties of the bonded phase.     

Study of solvation processes on cholesterol bonded phases

Four cholesterol bonded phases with different structures were investigated. The columns studied were packed with stationary phase containing cholesterol attached to the silica surface using different types of linkage molecules. The presence of the polar amino and carboxyl groups in the structure of the bonded ligand strongly influence on the solvation process. The possibility of hydrogen bonding, dipole-dipole and π-π electron interactions lead to preferential solvation of bonded ligands. The coverage density of bonded ligands and length of the linkage strongly influence the adsorption of solvent from the mobile phase. The removal of residual silanols during the hydrosilation procedure significantly influences solvation of the bonded phase. Excess isotherms of the commonly used solvents in RP HPLC (methanol and acetonitrile) were obtained using the minor disturbance method. For comparison of the stationary phases prepared on different silica gels the excess adsorbed amounts were calculated per volume of the stationary phase in the column. The hydrosilated UDC Cholesterol bonded phase is preferentially solvated by methanol whereas the highest coverage Cosmosil Cholester phase exhibit high adsorption of acetonitrile. Polar groups in the Amino-cholesterol type bonded phase are solvated with both solvent but the mechanisms of these processes are different.     

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 %.     

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.     

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.     

Solvent Effects in Reversed Phase Liquid Chromatooraphy for High and Low Loadings of Octadecylsilane on Microparticle Silica. I

Abstract

The selectivity of two ODS bonded phase packings, one with a high carbon load, the other with a low carbon load, were investigated. Many organic solvent/water mobile phases were used with three sample mixtures of varying polarity. When the separations were normalized, trends in the solubility parameters of the various classes of organic solvents were noted. Analyzing the separation factors and considering possible mobile phase, solute, and bonded phase interactions furnished some insight into possible separation/selectivity mechanisms.     

The effect of chain length of bonded organic phases in reversed phase high performance liquid chromatography

Summary Bonded hydrocarbon phases have been prepared by the reaction of organotrichlorosilanes with silica (Lichrosorb Si-100, 10 m, Merck) using conditions for maximum coverage. Alkyl phases, CH₃ (CH₂)_n-With n=7, 10, 12, 14, 17 and 20 and arylakyl phases, Ph (CH₂)_n—with n=0,2,4,6 were investigated. In reverse phase chromatography using water/methanol phases a linear relationship was between the capacity ratios k and the amount of coverage, independent of chain length. The selectivity, however, depends on the chain length of the bonded phase and molecular structure of the solute, whereas for halogenated benzene derivatives the selectivity is constant, and changing the mobile phase composition shows only a minor effect. The selectivity of 4,4-dibromodiphenyl in relation to the benzene derivatives is strongly influenced by the chain length and solvent composition. The utilization of chemically bonded organic long chain phases with maximum coverage makes it possible to reduce the water content in the water/methanol phase increasing the efficiency and loading capacity. The extent of maximum coverage was slightly dependent on the chain length and showed only a 10% decrease from n-octyl-to heneicosyl phase. Treatment of the bonded organic phase with TMCS was not effective with long chain materials, but further reaction occurred with bonded phenyl phase, which could be shown by IR-analysis.     

Cholesterol bonded phase as a separation medium in liquid chromatography. Evaluation of properties and applications

An extensive survey of the properties and separation capabilities of a cholesterol bonded phase is reported. The intermediate hydrophobic/hydrophilic properties of the bonded cholesterol material allows this stationary phase to be used for both reversed-phase and aqueous normal-phase separations. Interesting high selectivity is reported for the structural isomers of some antibiotics. The cholesterol bonded material does not display "phase collapse" in high aqueous content mobile phases. Variable temperature studies demonstrate that substantial structural changes of the bonded moiety occur that might be used to control selectivity. Finally, separation of some enantiomers of compounds with a variety of chemical structures is reported under reversed-phase conditions indicating that the cholesterol material may be chiral stationary phase with a broad range of applicability.     

Solvation processes on phenyl‐bonded stationary phases—The influence of polar functional groups

A series of phenyl‐bonded stationary phases with incorporated polar functional groups was subjected to an adsorption investigation. Measurement of acetonitrile and methanol adsorption was obtained using the minor disturbance method. It was observed that adsorption of organic solvent strongly depends on the presence of polar functional groups in the bonded phases that influence the hydrophobicity and polarity of the stationary phase surface. Additionally, relative adsorption of acetonitrile and methanol confirms earlier observations, that the presence of amine and amide groups in the stationary phase changes the relative elution strength of organic solvents. The heterogeneous surface of the stationary phase makes it possible to observe the competitiveness of the water and organic solvent adsorption.     

Selectivity of calixarene‐bonded silica phases in HPLC: Description of special characteristics with a multiple term linear equation at different methanol concentrations

Retention and selectivity characteristics of different calixarene‐, resorcinarene‐ and alkyl‐bonded stationary phases are examined by analyzing a set of test solutes covering the main interactions (hydrophobic, steric, ionic, polar) that apply in HPLC. Therefore Dolan and Snyder's multiple term linear equation has been adapted to fit the properties of calixarene‐bonded columns. The obtained parameters are used to describe retention and selectivity of the novel Caltrex^® phases and to elucidate underlying mechanisms of retention. Here, differences of stationary phase characteristics at different methanol concentrations in the mobile phases are examined. Both selectivity and retention were found to depend on the methanol content. Differences of these dependencies were found for different stationary phases and interactions. The differences between common alkyl‐bonded and novel calixarene‐bonded phases increase with increasing methanol content.     

Temperature dependence of equivalent chain length values on capillary columns of different polarity

The influence of temperature and capillary column stationary phase polarity on the equivalent chain length (ECL) values of unsaturated fatty acid methyl esters (FAMEs) is discussed. Comparisons are made of a bonded, nonpolar methyl silicone, bonded and nonbonded polyethylene glycols, and a highly polar, stabilized cyanosilicone stationary phase. The change in the ECL values over a 20° temperature range is used to demonstrate selectivity shifts and the influence of temperature on the separation of FAMEs on these phases. The effect of the degree of unsaturation of the FAME components, on the various stationary phases is also investigated.     

Comparison of four cholesterol‐based stationary phases for the separation of steroid hormones

The chromatographic behavior of steroid hormones on four cholesterol‐bonded stationary phases with different structures in binary methanol/water mobile phases was studied. Of the stationary phases tested, the commercially available stationary phases Cogent UDC cholesterol? and COSMOSIL cholester? provided better separations of steroid hormones in comparison to homemade aminocholesterol and diaminocholesterol stationary phases. The results show that the temperature has a significant influence on the retention and selectivity for steroid hormones separation. The temperature increase may cause changes in the elution order. From the dependences of the retention (ln k) on temperature (1/T), the standard partial molar enthalpy and standard partial molar entropy were calculated and their enthalpic and entropic contributions to the retention were compared. The enthalpic effects principally control the retention mechanism.     

Reversed-phase separation of basic tricyclic antidepressants using buffered and fluoroform-enhanced fluidity liquid mobile phases

In an effort to expand the range of applications of enhanced-fluidity liquid chromatography (EFLC) to strongly polar and basic analytes, fluoroform (CHF3) was investigated as a fluidity-enhancing agent. Fluoroform was chosen due to its high polarity, low viscosity and chemical inertness toward water and basic analytes. A group of representative basic compounds, tricyclic antidepressants, covering a wide range of polarity was chosen as model compounds. Their retention behavior on a C18 stationary phase in methanol/phosphate buffer and methanol/phosphate buffer/CHF3 mobile phases was characterized. The chromatographic performance with mobile phase conditions of different pH, with and without CHF3 addition and with addition of triethylamine was studied. The advantages of using CHF3 enhanced and buffered mobile phases were shown in the much improved chromatographic performance, such as shortened analysis time, increased efficiency, lower pressure drop and improved selectivity. Furthermore, this study demonstrated for the first time, that a commercial instrument could be readily utilized for EFLC separations which greatly expands the application range of the EFLC technique and chromatographic instrumentation.     

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.     

Influence of stationary phase solvation on shape selectivity and retention in reversed-phase liquid chromatography

In the past few decades, shape selectivity has drawn a great deal of attention from chromatographers. The chemistry and characteristics of bonded stationary phases such as phase type, length of bonded phase, surface coverage, and silica surface material have an effect on the shape selectivity of the columns. Although the effects of bonded phase shape selectivity are relatively well understood, one remaining question is the effect of intercalated solvent on shape selectivity. The intercalation of organic modifier and water molecules into the stationary phase is believed to introduce more rigidity into bonded alkyl chains in RPLC. The use of gas chromatography (GC) opens a new dimension to approach this question. C18 columns 4 cm in length were prepared in our laboratory and used in both LC and GC experiments. Shape selectivity and thermodynamic constants for the transfer of a solute from the mobile phase to the stationary phase have been determined as a function of monomeric octadecyl stationary phase bonding densities over the range of 1.44-3.43 micromol/m2 and a polymeric phase (nominal surface coverage 4.77 micromol/m2). Comparing LC and GC experiments, we observed: (a) similar relationships between shape and phenyl selectivities with monomerically bonded C18 phase densities; (b) different correlation of thermodynamic quantities (DeltaH degrees , DeltaS degrees , and DeltaG degrees ) versus bonded phase densities. The effects of high temperature and residual silanol groups are sources of difficulty in elucidation of the intercalated mobile phase role in selectivity and retention for GC measurements.     

Synthesis and evaluation of silica hydride‐based fluorinated stationary phases

Two novel silica hydride‐based fluorinated bonded phases have been synthesized using a hydrosilation procedure to test combined fluorine and hydride selectivity. The bonded moieties were characterized by elemental and spectral analysis. Chromatographic testing was done using hydrophilic analytes in the aqueous normal phase mode. At higher amounts of the nonpolar solvent in the mobile phase, there should be increased retention for solutes such as acids, bases and other polar compounds, whereas nonpolar solutes can be retained when water is increased as in RP chromatography. The synergistic effects of the fluorinated phase selectivity and aqueous normal phase retention on a hydride surface have been explored for small polar molecules. The stability and repeatability of the hydride‐based fluorinated stationary phases were evaluated. The use of acetone as the organic component in the mobile phase was also tested.     

High speed capillary GC on 10 m × 100 μm i.d. FSOT Columns

The chromatographic performance of 10 m × 100 μm i.d. capillary columns, coated with different stationary phases, was studied. TZ-ū plots were measured at different temperature program rates. The polarity and selectivity are strongly influenced by the temperature program rate. The analysis time depends on the capacity ratio and the selectivity factor; stationary phase selection thus is an important aspect in high speed capillary GC. A number of applications is presented and the applicability of split injection is discussed.     

Raman spectroscopy of octadecylsilane stationary phase conformational order. Effect of solvent

The effect of solvent on the conformation of alkyl chains of two octadecysilane-based stationary phases is probed using Raman spectroscopy. Spectral data indicate that the alkyl chains of commercially available polymeric and monomeric solid-phase extraction stationary phases are disordered to a varying extent by solvents of different polarity. For the polymeric octadecylsilane stationary phase, the polar solvents water, acetonitrile, methanol, acetone and isopropanol have little impact on the conformational order of the octadecylsilane bonded phase relative to air. However, the alkyl portion of this stationary phase is substantially disordered in the low-polarity solvents tetrahydrofuran, chloroform, benzene, toluene and hexane. The monomeric octadecylsilane stationary phase is less susceptible to disordering by solvents, although more disorder in the less polar solvents is also observed for this system. These results are interpreted in terms of the local surface bonding density and interchain spacing of these two stationary phases, and the ability of the solvent to penetrate the chains as a function of polarity. The results clearly demonstrate the ability of Raman spectroscopy to precisely indicate subtle changes in conformational order of alkylsilane stationary phases.     

An attempt directed toward enhanced shape selectivity in reversed-phase liquid chromatography: preparation of the dodecylaminated beta-cyclodextrin-bonded phase.

With the aim of preparing a stationary phase with a high shape-recognition ability for liquid chromatography, a new bonded phase was synthesized by coupling multiply dodecylamino-substituted beta-cyclodextrin (beta-CD) to 3-glycidoxypropyl-derivatized silica gel. The stationary phase prepared in this way was expected to have increased shape selectivity compared with that of conventional reversed-phase materials, due to solute interactions with the alkyl chain piles built up on the beta-CDs bonded to silica. The separation characteristics of the bonded phase were investigated using polycyclic aromatic hydrocarbons (PAHs) with different molecular shapes and compared with those of monomeric ODS and native beta-cyclodextrin-bonded phases. The newly developed stationary phase was found to be highly selective for PAHs.