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.     

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.     

Isocratic Separation of Dansylated Biogenic Amines on a C8‐Bonded Silica Column under the LC Elution of Methanol‐Based Mobile Phase

Under the elution of methanol‐based mobile phase, the isocratic resolution of 12 biogenic amines, including 1 aromatic, 2 heterocyclic and 9 aliphatic amines, as the dansylated derivatives has been accomplished in less than 25 minutes on a 15 cm C₈‐bonded column. The resolution can not be reproduced on other examined alkyl‐bonded phases (e.g., C₄ and C₁₈) under the same chromatographic conditions, or in the reversed‐phase mode. The retention, mainly as a result of hydrophobic interaction between analyte and stationary phase, can be adjusted by varying the percentage of methanol in the mobile phase. Also, incorporating acetic acid as additive to the mobile phase to protonate the analyte and silanol groups that are little shielding on the surface of silica gel reduces the dipole‐dipole interaction, and thus the retention scale, which in turn deteriorates the resolution. Furthermore, the elution reversal is plausible for some of analytes as a greater percent of acetic acid is used in the elution. Values of correlation coefficients (R²) range between 0.9995 and 0.9996, indicating good linearity.     

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 %.     

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.     

Partially Methylated β-Cyclodextrin Analysis: A Systematic Approach to Appropriate RP Column Selection

In order to establish guidelines to help the analyst in the choice of the most suitable octadecyl or octyl bonded phase for the LC analysis of a given partially methylated β-CD sample, analyses of four commercially available dimethyl-β-cyclodextrins (DM-β-CDs) have been carried out on nine octyl (C₈) or octadecyl (C₁₈) silica bonded or polymeric bonded phases which differ significantly in their hydrophilic and hydrophobic properties. Chromatograms show that the nature of the packing materials has considerable influence on the resolution of complex mixtures composed of closely related compounds such as partially methylated β-CDs. Among various kinds of C₈ and C₁₈ bonded phases, silica based and monomeric phases which present both reinforced hydrophobic and polar interactions showed the best performance. Whatever the complexity of the commercial DM-β-CD, the richest chromatographic fingerprints, which best depict the complexity of the mixture, are obtained with Nucleosil 50-5-C₈ column. For the simplest mixtures, Nucleosil 50-5-C₈ column with acetonitrile-water (34:66) as mobile phase is the most suitable chromatographic system and leads to the best resolution between heptakis (2,6-di-O-methyl)-β-CD and hexakis (2,6-di-O-methyl)-mono(2,3,6-tri-O-methyl)-β-CD (14 OCH₃ and 15 OCH₃). This chromatographic system might enable an LC-MS coupling for direct identification of the different components in the mixture as well as control of batch to batch variations.     

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.

     

Poly(carboxyethyl acrylate-co-ethylene glycol dimethacrylate) precursor monolith with bonded octadecyl ligands for use in reversed-phase capillary electrochromatography

A carboxy precursor monolithic column, namely poly(carboxy ethyl acrylate-co-ethylene glycol dimethacrylate) was first produced in a 100 μm i.d. fused-silica capillary and subsequently surface bonded with n-octadecyl (C₁₈) ligands by a post-polymerization functionalization process with octadecylamine in the presence of N,N´-dicyclohexylcarbodiimide. The bonding of octadecyl ligands was achieved via an amide linkage between the carboxy functions of the precursor monolith and the amino group of the octadecylamine compound. The resulting C₁₈ monolith exhibited a very low electroosmotic flow (EOF), a fact that required the incorporation of small amounts of 2-acrylamido-2-methylpropane sulfonic acid (AMPS) in the polymerization solution to produce a precursor monolith with fixed negative charges of sulfonate groups. This may indicate that the conjugation of the carboxy functions with octadecylamine occurred to a large extent so that the amount of residual carboxy functions was sparsely dispersed and not enough to produce a desirable EOF. The EOF velocity of the C₁₈ column having fixed negative charges provided by the incorporated AMPS increased with increasing ACN content of the mobile phase signaling an increased binding of mobile phase ions to the polar amide linkages near the monolithic surface, and a decreased viscosity of the mobile phase, both of which would result in increased EOF velocity. The C₁₈ monolithic column constituted a novel nonpolar sorbent for reversed-phase capillary electrochromatography for nonpolar solutes, e.g., alkylbenzenes, alkylphenyl ketones, and polyaromatic hydrocarbons, and slightly polar compounds including phenol and chlorophenols. The C₁₈ monolithic column exhibited relatively high selectivity toward chlorophenols differing by one chloro substituent.     

Effect of the residual silanol group protection on the liquid chromatographic resolution of α‐amino acids and proton pump inhibitors on a ligand exchange chiral stationary phase

A new ligand exchange chiral stationary phase (new CSP) containing residual silanol group‐protecting n‐octyl groups on the silica surface was prepared by treating a ligand exchange CSP (original CSP) based on sodium N‐[(R)‐2‐hydroxy‐1‐phenylethyl]‐N‐undecylaminoacetate bonded to silica gel with excess n‐octyltriethoxysilane. The new and original CSPs containing an identical amount of chiral selector were applied to the resolution of α‐amino acids and proton pump inhibitors (PPIs) including omeprazole, pantoprazole, lansoprazole, and rabeprazole. The separation factors (α) and resolutions (R_S) were greater on the new CSP than on the original CSP except for the resolution of asparagine. The trends of the retention factors (k₁) for the resolution of α‐amino acids on the new and original CSPs with the variation of the organic modifier content in aqueous mobile phase were opposite to those for the resolution of PPIs. Removal of the nonenantioselective interactions between the residual silanol groups and the analytes and the improved lipophilicity of the new CSP were proposed to be responsible for the improved chiral recognition ability of the new CSP and the different retention behaviors of the enantiomers between the new and original CSPs.     

Protein separation on a polar-copolymerized C<Subscript>8</Subscript> stationary phase

A novel polar-copolymerized C₈ stationary phase composed of octyl and chloropropyl (C₈-C₃Cl)-bonded silica has been developed for protein separation. Separation of standard proteins on this homemade column was investigated and compared with separation on a Waters Symmetry 300 C₄ column. Results indicated that the chromatographic performance of the homemade column in the separation of proteins was excellent. The new polar copolymerized C₈ stationary phase enabled glycoprotein separation with good resolution and reproducibility. It was also used for purification of ovalbumin glycoprotein in order to improve identification of the protein.     

Electrochromatographic performance of conventional and polar-embedded C16 silica monolithic stationary phases

A novel monolithic silica column that has a polar‐embedded amide‐secondary amine group linking with C16 functionality for RP‐CEC is described. The amide‐secondary aminealkyloxysilane was synthesized by the reaction of 3‐(2‐aminoethylamino) propyltrimethoxysilane with hexadecanoyl chloride. Then, the silylant agent was bonded to the silica monolith matrix to produce hexadecanamide‐secondary amine bonded silica (HDAIS) monolithic column. The electrochromatographic performance of HDAIS monolithic column for the separation of neutral, basic and polar solutes was studied, which was compared to that using the hexadecyl bonded silica monolithic column. The HDAIS monolithic column displayed reduced hydrophobic retention characteristics in the separation of five hydrophobic n‐alkylbenzenes and four polar phenols when compared to the hexadecyl bonded silica monolithic column. A very much reduced silanol activity of HDAIS monolithic column was observed in the separation of test basic mixture including four aromatic amines, atenolol and metoprolol with 10 mM borate buffer (pH 7.5) containing 30% v/v ACN as the mobile phase. The comparison results indicate good performance for both polar and basic mixtures on HDAIS monolithic column in RP‐CEC, and also show promising results for further applications.     

Influence of a water vapor admixture in the carrier gas on capacity coefficients of polar organic compounds

The influence of a water vapor admixture in helium, nitrogen, and carbon dioxide on capacity coefficients of C₃−C₅ alcohols and pyridine during chromatography process in capillary columns with polar (PEG-20M) and nonpolar (SE-30) stationary phases was studied. The introduction of a water admixture into the carrier gas, increases the capacity coefficient of polar organic compounds on the capillary column with PEG-20M and has almost no effect on this value in the case of SE-30. The change in retention of polar organic compounds on the capillary column with the PEG-20M polar phase occurs due to a change in the properties of the stationary phase when it adsorbs water from the mobile phase. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 11, pp. 2258–2261, November, 1998.     

保留方程式和流动相体积

从溶质的保留方程式出发,讨论了液相色谱柱中流动相体积V_m和各种测量V_m方法. 借助乙腈在C₈键合相上的绝对吸附等温线,评价了这些测量方法的局限性.     

The stationary phase capacity concept in elution liquid chromatography

The stationary-phase capacity concepts derived from linear capacity are discussed in connection with the needs of analytical, trace enrichment analysis and preparative chromatography and shown to be unsuited to them. A new concept based on stationary-phase saturation and called “available capacity” is proposed. It generalizes the ion-exchanger exchange capacity to adsorption and partition chromatography when the sampling solvent is the mobile phase. In linear elution chromatography the available capacity is proportional to the solute concentration C_o and to the analytical capacity factor k′ for given C_o and k′ values, it is independent of the nature of the solute. Furthermore, when both the concentrations and the analytical capacity factors (practically, for C_o ≥ 1 M and k′ ≥ 10, respectively) are high, the available capacity reaches a value roughly independent of C_o and k′, called “maximum available capacity” and related only to the number of sites available on the stationary phase. Numerous measurements were made in ion-exchange, adsorption, and reversed-phase chromatography. For solutes having a single polar functional group interacting with the stationary phase, the orders of magnitude of the maximum available capacity are 1.2 mmole g^?1 for a classical silica gel (Partisil 5 μm, 400m^?2 g^?1 with a water content of 2.7%); 1.8 mmole g^?1 for the Lichroprep RP 8 octyl bonded silica (11.6% carbon content); 3.8 mmole g^?1 for an anion exchanger resin of Dowex type.     

Chromatographic Behaviour and Lipophilicity of Estradiol Derivatives

The retention behaviour of estradiol derivatives has been studied by HPLC on polar chemically bonded stationary phases: C₃CN, DIOL and C₃NH₂, commercially available columns. The mobile phases used were: methanol-water and acetonitrile-water in various proportions. Reversed-phase chromatography occurred on polar chemically bonded stationary phases. Correlation between the retention constants of estradiol derivatives obtained on polar chemically bonded phases and log P calculated via different methods was examined too.     

Structure–Property Relationship of Supramolecular Ferroelectric [H‐66dmbp][Hca] Accompanied by High Polarization,Competing Structural Phases,and Polymorphs

Three polymorphic forms of 6,6′‐dimethyl‐2,2′‐bipyridinium chloranilate crystals were characterized to understand the origin of polarization properties and the thermal stability of ferroelectricity. According to the temperature‐dependent permittivity, differential scanning calorimetry, and X‐ray diffraction, structural phase transitions were found in all polymorphs. Notably, the ferroelectric α‐form crystal, which has the longest hydrogen bond (2.95 Å) among the organic acid/base‐type supramolecular ferroelectrics, transformed from a polar structure (space group, P2₁) into an anti‐polar structure (space group, P2₁/c) at 378 K. The non‐ferroelectric β‐ and γ‐form crystals also exhibited structural rearrangements around hydrogen bonds. The hydrogen‐bonded geometry and ferroelectric properties were compared with other supramolecular ferroelectrics. A positive relationship between the phase‐transition temperature (T_C) and hydrogen‐bond length (<d>) was observed, and was attributed to the potential barrier height for proton off‐centering or order/disorder phenomena. The optimized spontaneous polarization (P_s) agreed well with the results of the first‐principles calculations, and could be amplified by separating the two equilibrium positions of protons with increasing <d>. These data consistently demonstrated that stretching <d> is a promising way to enhance the polarization performance and thermal stability of hydrogen‐bonded organic ferroelectrics.     

Evaluation of the Chromatographic Performance of Conventional, Polar-Endcapped and Calixarene-Bonded Stationary Phases for the Separation of Water-Soluble Vitamins

The performance of four different reversed-phase columns which included a conventional C₁₈ phase, a C₁₈ polar-endcapped phase, an ether-linked phenyl polar-endcapped phase and a calixarene-bonded phase has been systematically compared for the separation of mixture of some water-soluble vitamins containing basic, neutral and acidic compounds of different polarities, as well as different functional groups at three pH levels and different proportions of buffer/methanol. The characteristics of water-soluble vitamins make this combination of compounds very useful as a test mixture to check column performance with real samples. Due to the physical and chemical differences between these compounds, the type of chosen column has a significant influence on the chromatographic behavior. Results of this comparison show that the C₁₈ polar-endcapped phase was the most suitable for the separation of this group of vitamins. The presence of a polar group as an endcapping agent does not seem to influence the overall hydrophobic nature of the polar-endcapped stationary phases. At the same time, these phases displayed enhanced hydrogen bonding and silanol activity.     

Sol–gel coatings with covalently attached methyl,octyl, and octadecyl ligands for capillary microextraction. Effects of alkyl chain length and sol–gel precursor concentration on extraction behavior

Fused silica capillaries with surface-bonded sol–gel coatings containing covalently attached octadecyl, octyl, and methyl groups were prepared for capillary microextraction (CME) hyphenated on-line with high-performance liquid chromatography (HPLC). For this, octadecyltrimethoxysilane (C₁₈TMS), octyltrimethoxysilane (C₈TMS), or methyltrimethoxysilane (MTMS) was used as the respective sol–gel precursor. Hydrolytic polycondensation of these precursors led to the formation of surface-bonded sol–gel sorbents with pendant alkyl groups ready to serve as the extraction medium; no additional surface derivatization reactions were needed to anchor these ligands to the surface. Extraction behaviors of two sets of microextraction capillaries with alkyl-bonded sol–gel coatings were investigated: (a) capillaries prepared with a constant molar concentration of these precursors in the sol solution, and (b) capillaries prepared with varied molar concentrations of C₈TMS in the sol solution. Among the capillaries prepared using sol solutions with the same molar concentration of sol–gel precursor, the detection limits for nonpolar and polar analytes ranged from 0.3 ng/L to 213.9 ng/L. The sol–gel octadecyl-coated capillaries were found to be the most efficient at extracting these analytes, followed by the sol–gel octyl-coated capillaries, followed by the sol–gel methyl-coated capillaries. The results of this study point to the possibility that polar analytes are extracted through synergistic molecular level interactions of the polar and nonpolar parts of the analyte molecules with the alkyl chains and silanol groups within the sol–gel coatings. These coatings also demonstrated run-to-run and capillary-to-capillary reproducibility, with HPLC peak area RSD values ranging from 1.1% to 9.6% and 1.3% to 10.0%, respectively. In the set of sol–gel octyl capillaries with varied molar concentrations, the capillaries prepared with 0.514 M concentration of C₈TMS in the sol solution were most efficient in extracting nonpolar and polar analytes. When higher or lower concentrations of C₈TMS were used in the sol solution, the resulting sol–gel coated capillaries were less efficient in extracting nonpolar and polar analytes.     

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.     

Synthesis of fluorosilicone having highly fluorinated alkyl side chains based on the hydrosilylation of fluorinated olefins with polyhydromethylsiloxane

Hydrosilylation of fluorinated olefins with polyhydromethylsiloxane (PHMS) in the presence of a platinum catalyst was investigated to synthesize fluorosilicone having highly fluorinated alkyl side chains (R_f; C_nF_2n+1? ). The hydrosilylation of 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10‐heptadecafluoro‐1‐decene (C₈F₁₇CH?CH₂) ( 1 ) with poly(dimethylsiloxane‐co‐hydromethylsiloxane) {(CH₃)₃SiO[? (H)CH₃SiO? ]₈[? (CH₃)₂ SiO? ]₁₈Si(CH₃)₃} ( 4 ) converted the hydrogen bonded to silicons into the 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10‐heptadecafluorodecyl group or fluorine bonded to silicons in the ratio of about 52:48, and the formation of the byproduct C₇F₁₅CF?CHCH₃ ( 8 ) was observed. The hydrosilylation of 7,7,8,8,9,9,10,10,11,11,12,12,13,13,14,14,14‐heptadecafluoro‐4‐oxa‐1‐tetradecene (C₈F₁₇CH₂CH₂OCH₂CH?CH₂) ( 2 ) with 4 converted the hydrogen bonded to silicons into the 7,7,8,8,9,9,10,10,11,11,12,12,13,13,14,14,14‐heptadecafluoro‐4‐oxa‐tetradocyl group bonded to silicons, but an excess amount of 2 was required to complete the reaction because the isomerization of 2 occurred in part to form C₈F₁₇CH₂CH₂OCH?CHCH₃ ( 9 ). The hydrosilylation of 4,4,5,5,6,6,7,7,8,8,9,9, 10,10,11,11,11‐heptadecafluoro‐1‐undecene (C₈F₁₇CH₂CH?CH₂) ( 3 ) with 4 converted the hydrogen bonded to silicons into the 4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11‐heptadecafluoroundecyl group bonded to silicons. This type of fluorinated olefin was successfully applied to the hydrosilylation with other PHMS's that involved a homopolymer of PHMS and a cyclic PHMS. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 3120–3128, 2002