CP/MAS NMR investigations of silica gel surfaces modified with aminopropylsilane

Summary Aminopropyl chemically bonded phases for high performance liquid chromatography (HPLC) have been prepared using mono- and trifunctional methoxyor ethoxysilanes. Three types of silica gel with different surface characteristics were used as support for the chemically bonded phases (CBPs). Surface characteristics of the packings before and after chemical modification were determined by porosity parameters, elemental analysis and CP/MAS NMR spectroscopy.²⁹Si and¹³C CP/MAS NMR investigations gave informations about different interactions between aminosilyl ligands and/or these ligands and/or water molecules condensed in the pores of the silica gel surface. With decreasing pore diameter of the silica gel the proportion of protonated aminopropyl ligand increases.     

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.     

Influence of the Structure of Chemically Bonded C 18 Phases on HPLC Separation of Naproxen Glucuronide Diastereoisomers

Abstract

On the basis of Nucleosil-100 a series of materials with varying structure and different coverage density of chemically bonded C₁₈ phases (CBP) were prepared. The physico-chemical characteristics of these packings e.g. porosity and carbon content were studied by the BET method and CHN analysis. The structure of the C₁₈ CBP was determined by solid state CP/MAS NMR. The prepared packings and columns have been applied for HPLC separation of naproxen and its two diastereoisomeric conjugates with glucuronic acid. Material with monomeric C₁₈ CBP structure and with high coverage density has given good and reproducible separation results.     

Comparison of D,L-Mandelic Acid Resolution on Zeolite and Silica Supported Pirkle-Type Chiral Stationary Phases

 Zeolite A, a material of crystalline character, and Hypersil silica have been used as support for the preparation of chiral stationary phases. On the amorphous silica support surface the silanol groups are randomly dispersed. The crystalline zeolite secondary building units consisting primarily of SiO₄, AlO₄ ⁻ tetrahedra determine the regularity of surface silanol groups. Owing to the crystal lattice structure, the location of silanols is well determined and hence the dispersion of chiral selector molecules chemically bonded onto the zeolite surface silanol groups is fundamentally arranged. Amides of DNB-L-Leu, DNB-L-Phe, B-L-Leu chiral selector molecules were anchored onto the zeolite silanols and B-L-Leu onto the silica support silanols. Lipophilic buffer in RP conditions has dynamically modified the residual silanols of each support. The enantioseparation of ion paired D,L-mandelic acid from aqueous solution on the zeolite and silica supported chiral stationary phases prove a superior enantioseparation on the zeolite supported phases. Revision February 18, 2000.     

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 %.     

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.     

Application of bromoacetate‐substituted β‐CD‐bonded silica particles as chiral stationary phase for HPLC

Bromoacetate‐substituted [3‐(2‐O‐β‐cyclodextrin)‐2‐hydroxypropoxy]propylsilyl‐appended silica particles (BACD‐HPS), an important and useful synthetic intermediate for preparation of novel types of macrocycles‐capped β‐CD‐bonded silica particles including crown ether/cyclam/calix[4]arene‐capped β‐CD‐bonded silica particles, have been prepared and used as chiral stationary phase for HPLC. This synthetic stationary phase is characterized by means of elemental analysis. For the first time, the chromatographic behavior of BACD‐HPS was systematically evaluated with several disubstituted benzenes and some chiral drug compounds under both normal and RP conditions in HPLC. The results show that BACD‐HPS has excellent selectivity for the separation of aromatic positional isomers and chiral isomers of some drug compounds when used as stationary phase in HPLC.     

Quick and sensitive HPLC separations on non-porous reversed-phase packings

Summary The aim of this work was to evaluate reversed-phase chemically bonded non-porous (micropellicular)d _p=1.5 μm stationary phases. On these modern phases the time for analysis of complex mixtures of solutes—whether monomeric or polymeric (e.g. drugs, vitamins, peptides, or protein)—is very short compared with that on porous phases. Different surface chemistries were elaborated for the separation of different types of sample. For the separation of small molecules a long-chain (C₁₄) hydrocarbon-coated phase seems to be optimum; a short chain (C₆) hydrocarbon bonded to the surface of the silica seems better for the separation of polymers. The efficiency, the low analysis times, and sensivities were demonstrated by separation of different proteins, peptides, drugs, alkaloids, and mixtures of water and fat-soluble vitamins.     

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.     

Synthesis of chemically bonded squalene(ane) phases for microcolumn gas chromatographic separation of low-molecular-weight hydrocarbons in extraterrestrial atmospheres

Summary Chemically bonded squalene(ene) phases have been synthesized via a hydride modified silanization process using dimethylchlorosilane, triethoxysilane and trichlorosilane. The surface structure of the bonded phases were characterized with DRIFT and solid state NMR studies. The results of the latter indicate successful bonding of a non-terminal olefin to the silica surface and that bonding of individual squalene molecules appeared not to occur at the same site(s) presumably due to steric hindrance. Among the phases examined, the trichlorosilane-based material exhibits better chromatographic properties probably due to greater surface coverage and hence effective non-polar interaction between the solutes and the bonded ligands. Under isothermal conditions, the present packings produced fast and efficient separation of C₁–C₄ saturated hydrocarbons on shorter columns than similar columns containing alkyl- or bidentate alkyl-modified silica packings at low column head pressure and are thermally stable at temperatures up to 250°C or higher.     

Performance of zwitterionic hydrophilic interaction LC for the determination of iodinated X‐ray contrast agents

This study compares the separation performance of a group of iodinated X‐ray contrast media on four different columns. The first three were two stationary phases (SPs) modified with C₁₈ and a polar‐embedded SP (polar amide group bonded to an alkyl chain), all of which worked under RP‐LC mode. The fourth was a zwitterionic sulphoalkylbetaine SP, working under the hydrophilic interaction LC (HILIC) mode. After the optimisation of the different parameters, the zwitterionic column displayed the best separation, which also overcomes the problems encountered when these analytes were separated under RP‐LC. Moreover, when HILIC is coupled to MS/MS, sensitivity is enhanced. However, when sewage samples were analysed by SPE followed by the optimal HILIC–MS/MS, the sensitivity of the method was affected due to the high matrix effect, which had to be solved by dilution of the extract. Finally, the method was preliminarily validated with sewage and the figures of merit were comparable to those of the SPE–RP‐LC–MS/MS.     

An integrated coupled column liquid chromatography system for the determination of leukotriene metabolites in biological samples

Summary A fully integrated chromatographic system was developed for the determination of leukotrienes in biological samples using photodiode-array detection (PDAD), which eliminates time consuming manual sample handling steps. A special solid phase extraction, (SPE) methodology for leucotriene metabolite stability was developed which increased the recoveries and eliminates the contamination risk of biological samples. The inherent instability (autooxidation) of many of the leukotriene mediators, and the adsorption effects onto exposed surfaces in vials and in the chromatographic system were found to be very important parameters to control in order to circumvent high loss of sample analytes. By binding the cell supernatants to the functionalities of the SPE support stabilised these mediators. Cell culture samples were eluted through a disposable C₁₈ SPE column. The SPE columns were allowed to thaw and deposited in an automated sample handling unit (ASPEC XL). Desorption of the analytes was followed by a second on-line SPE step, to eliminate remaining interfering matrix compounds. Typical recoveries when stored at −70°C were in-between 55–97% except for (LTE₄) which was found to be around 40% after 72 days of storage. Seven reversed-phase packings were studied. Selectivity factors, as well as the separation efficiencies, were found to differ for the various C₁₈ bonded silica stationary phases. This integrated on-line column liquid chromatographic system was applied to the determination of leukotriene B₄, leukotriene C₄, leukotriene D₄, leukotriene and E₄ in human cell extracts using prostaglandin B₂ as the internal standard. More than 1500 biological samples were analysed. Some validation data are presented for unattended operations.     

Chiral separation of amides of amino acid on a (S)‐N‐(3,5‐dinitrobenzoyl)leucine‐N‐phenyl‐N‐propylamide‐bonded silica using nonaqueous capillary electrochromatography

(S)‐N‐(3,5‐dinitrobenzoyl)leucine‐N‐phenyl‐N‐propylamine‐bonded silica was used as a chiral stationary phase for separation of a set of racemic π‐acidic and π‐basic α‐amino acid amides in electrolyteless ACN‐water eluents by CEC in the RP and polar organic (PO) modes. The effect of the amount of water in the ACN‐water eluent on chiral separation was examined. As water is added to ACN, retention was shortened but resolution and selectivity deteriorated severely. Retention, enantioselectivity, and resolution factors obtained in 100% ACN were compared with those in an n‐hexane‐isopropanol eluent with a small amount of water by normal phase (NP) CEC. Much shorter retention times with comparable enantioselectivities were observed with 100% ACN, demonstrating the advantage of separation on (S)‐N‐(DNB)leucine‐N‐phenyl‐N‐propylamine‐bonded silica in PO‐CEC over NP‐CEC.     

A novel method for the preparation of C18 ester-bonded RP-HPLC packings

Summary A novel procedure had been developed for the preparation of RP packings. A C₁₈ alkyl-chain was bound onto spherical silica (particle size d_p=5 μm, s=300 m²g⁻¹) with glycidoxypropyltrimethoxysilane as coupling agent. Elemental analysis, X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FT-IR) and chromatographic evaluation confirmed the bonding of the packings and their RP behavior.     

C18, C8, and perfluoro reversed phases on diamond for solid-phase extraction

In spite of advances in solid-phase extraction (SPE) technology there are certain disadvantages to current SPE silica-based, column packings. The pH range over which extraction can occur is limited and each column is generally only used once. New diamond-based reversed SPE phases (C₁₈, C₈, and perfluorinated) were developed in our laboratories. Studies were done which show that these phases do not have the same limitations as traditional silica-based stationary phases. The synthesis and properties of these diamond-based phases are presented, and the stability, percent recovery, and column capacity are given for the C₁₈ phase.     

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.

     

Ganoderma species discrimination by dual-mode chromatographic fingerprinting: A study on stationary phase effects in hydrophilic interaction chromatography and reduction of sample misclassification rate by additional use of reversed-phase chromatography

Acetonitrile–water extracts of several Ganoderma species – a mushroom being used in Traditional Chinese Medicine – were analysed by liquid chromatography–UV detection in hydrophilic interaction chromatography (HILIC) and reversed-phase (RP) elution modes. A set of six polar stationary phases was used for HILIC runs. These columns had remarkably different separation properties under binary gradient conditions as evinced by hierarchical cluster analysis on retention patterns of seven test compounds. Complementary measurements of RP chromatograms were carried out on a C₁₈ packing. Injection precision (n = 5) and intra-day precision (n = 5) were each <2.0% RSD (HILIC) and <0.7% RSD (RP) for relative retention times of main characteristic peaks of a sample extract while for relative peak areas RSD values were max. 6.8%. Repetitive analysis (n = 7) of a processed sample stored in the autosampler tray for 48 h was used to confirm within-sequence sample stability. Eleven Ganoderma lucidum samples served as training set for the construction of column-specific simulated mean chromatograms. Validation with twelve samples comprising G. lucidum, Ganoderma sinense, Ganoderma atrum, and Ganoderma tsugae by correlation coefficient based similarity evaluation of peak patterns showed that a discrimination of G. lucidum from other Ganoderma species by means of chromatographic fingerprints is conceptually possible on all columns, except of a bare silica packing. The importance of the combined use of RP and HILIC fingerprints to improve the rate of correct sample classification was demonstrated by the fact that each one G. sinense specimen was wrongly assigned being G. lucidum by all HILIC fingerprints but not the RP fingerprint and vice versa. The present data revealed that (i) the analysis of complex biological materials by quasi orthogonal chromatographic modes such as HILIC and RP may deliver more discriminative information than single-mode approaches which strengthens the reliability of fingerprint-based sample classification and (ii) different retention and selectivity characteristics of polar bonded silica packings in the HILIC elution mode may only have a minor impact on chemometric sample discrimination capabilities in such kind of pattern-oriented metabolomics separation problems.     

SPE for the simultaneous determination of various isothiocyanates

Several SPE sorbents were investigated for the extraction of a group of chemically diverse isothiocyanates (ITCs). They included bonded silica, carbon‐based, and polymer‐based sorbents with various functional groups. Results showed large differences in the ability of these sorbents to simultaneously extract ITCs from standard solutions. Recovery rates were on average the highest with divinylbenzene (DVB) based polymeric sorbents, especially with a DVB/N‐vinylpyrrolidone copolymer that had recovery rates ranging between 86.7 and 95.6%. These sorbents achieved the most balanced extraction efficiency between aliphatic and aromatic, polar, and nonpolar ITCs. With graphitized carbon, C₁₈‐bonded silica, and amide‐containing sorbent, recovery levels were higher for the two least polar aromatic ITCs (benzyl ITC and phenylethyl ITC), whereas for the polar aliphatic ITCs levels were the lowest. The least retained one, was methyl ITC that is the most polar with recoveries between 0 and 31.5%. The presence of amide groups, especially in a polyamide sorbent, appeared to be particularly unsuitable for the extraction of aliphatic ITCs. A copolymer made up of DVB and N‐vinylpyrrolidone was therefore shown to be the most suited for the extraction of both aliphatic and aromatic ITCs.     

Effect of Chiral Additives in the Preparation of Cellulose-Based Chiral Stationary Phases in HPLC, and Effect on Enantiomer Resolution

Chiral stationary phases (CSPs) for high-performance liquid chromatographic (HPLC) have been prepared by coating silica gel with cellulose tribenzoate or cellulose trisphenylcarbamate. The effect of chiral additives on preparation of the CSPs was studied with (+)-l-mandelic acid, (−)-2-phenyl-1-propanol, (+)-1-phenyl-1,2-ethanediol and (−)-1-(1-naphthyl)ethanol as chiral additives for cellulose tribenzoate and (−)-2-phenyl-1-propanol and (+)-phenylsuccinic acid as chiral additives for cellulose trisphenylcarbamate. The results showed that chiral recognition by these stationary phases was increased in comparison with the original CSPs, especially the resolution (R _S) obtained. The method can be used to improve the efficiency of enantiomer separation by silica gel stationary phases coated with polymers.     

Separation of 1,4-Dihydropyridine Derivative and Its Oxidized Form

Derivatives of 1,4-dihydropyridine (DHP) still play an important role in treatment of cardiovascular diseases. Typical degradation of the DHP ring is aromatization to pyridine ring which occurs both chemically and biochemically. It is, therefore, important to have a reliable and robust analytical method for separation of DHPs from their oxidized counterparts. Separation of closely-related substances possessing similar hydrophobicity, such as DHP and its oxidized form, can be challenging on conventional alkyl-bonded sorbents. In this study, an impact of reversed-phase (RP) liquid chromatography conditions on separation of the DHP/Ox pair has been investigated. Initially, a systematic study has been performed on 33 commercial RP columns with mobile phase acetonitrile/water for separation of foridone and its corresponding oxidized form. The retention and selectivity are discussed in view of the hydrophobic-subtraction model. Best separation was found replacing conventional C₁₈ sorbents with ones containing an embedded polar group due to polar interactions. Similarly, application of cyano columns resulted in efficient separation of analytes. Organic modifier of mobile phase (methanol vs. acetonitrile) contributed significantly to separation of foridone from its oxidized counterpart. Separation of six chemically diverse DHPs from corresponding oxidized forms was studied on seven RP columns (traditional C₁₈ sorbent, alkyl sorbent with polar embedded group, two different aromatic phases, pentafluorophenylpropyl sorbent and sorbent with straight chain perfluorohexyl ligand). Both acetonitrile and methanol were applied as organic modifier. It was found that application of alkyl sorbent with an embedded polar group (column Zorbax Bonus RP) or cyano sorbent (column ACE CN) yields clear separation of chemically diverse DHPs from their oxidized forms.