Evaluation of a Doubly Zirconized Silica-Based Stationary Phase for HPLC

This paper describes the preparation, characterization, and application of a chemically bonded and endcapped C18 stationary phase having a doubly zirconized silica support. The stationary phase was characterized using infrared and nuclear magnetic resonance (¹³C and ²⁹Si) spectroscopies, elemental analysis, and surface and thermogravimetric determinations, and evaluated chromatographically using several test mixtures, indicating acceptable efficiency, and asymmetry. The new phase was used for several different applications including the determination of the possible presence of six pesticides in orange juice, using a modified QuEChERS procedure for extraction.

     

Polar-copolymerized approach based on horizontal polymerization on silica surface for preparation of polar-modified stationary phases

A new approach for preparation of polar-modified reversed-phase liquid chromatography stationary phases was developed by using horizontal polymerization technique on silica surface, which was defined as “polar-copolymerized” approach. Based on this new approach, a representative polar-copolymerized stationary phase composed of mixed n-octadecyl and chloropropyl (C18–C3Cl) was synthesized. The resulting stationary phase named C18HCE was characterized with elemental analysis and solid phase ¹³C and ²⁹Si NMR, which proved the chemistry of polar-copolymerized stationary phases. Chromatographic evaluation and application of the C18HCE were also investigated. The results of preliminary chromatographic evaluation demonstrated that the C18HCE stationary phase exhibited 100% aqueous mobile phase compatibility, low silanol activity. In addition, the application results demonstrated that the C18HCE had superior separation performance in alkaloids separation at acidic conditions compared to some commercial stationary phases.     

Preparation and Evaluation of P-tert-Butyl-calix[8]arene-bonded Silica Stationary Phase for High Performance Liquid Chromatography

ABSTRACT

A new p-fert-butyl-calix[8]arene-bonded silica gel stationary phase was synthesized through heterogeneous functionalisation of suspended porous silica. A characterization of its structure was carried out by using elemental analysis, FTIR and ¹³C solid state NMR spectroscopy. Chromatographic performance of the new packing material was investigated by employing polycyclic aromatic hydrocarbons (PAHs) as probes and using methanol-water as mobile phase. The investigations show that the new stationary phase behaves as a reversed phase stationary phase. The liquid chromatographic separation of PAHs solutes on the new bonded phase was compared with that on a p-tert-butyl-calix[4]arene-bonded silica stationary phase. The new p-tert-butyl-calix[8]arene-bonded phase exhibited higher retention and better separation selectivity, although the carbon content and coverage of the new packing material was lower than that of the p-tert-butyl-calix[4]arene bonded silica stationary phase. A possible retention mechanism for the new packing material was also proposed.     

Stationary Phases 21: A New Polar Substituted Triazine Stationary Phase for HPLC

A method for preparing a new polar substituted triazine stationary phase is described. The structure of the triazine phase on silica was characterized by elemental analysis, and by FTIR, solid state FT-¹³C NMR, and ²⁹Si NMR spectral analysis. The chromatographic properties of this packing material have been evaluated by using a number of different solutes, and the properties compared with those of a commercial stationary phase RP-18. It is found that this triazine phase has weak π-donor ligands on the silica surface.     

Temperature programmed retention indices: Calculation from isothermal data. Part 2: Results with nonpolar columns

The procedure for calculating linear temperature programmed indices as described in part 1 has been evaluated using five different nonpolar columns, with OV-1 as the stationary phase. For fourty-three different solutes covering five different classes of components, including n-alkanes and alkyl-aromatic compounds, both isothermal and temperature programmed indices were determined. The isothermal information was used to calculate temperature programmed indices. For several linear programmed conditions accuracies better than 0.51^T-units were usually obtained. The results are compared with published procedures. It is demonstrated that isothermal retention information obtained on one column can be transferred to another column with the same stationary phase but different column dimensions and/or phase ratio. The temperature programmed indices calculated in this way also have an accuracy better than 0.51^T-u. The temperature accuracy and precision of the GC-instrumentation used was of the order of 0.1°C. All calculations can be run with a Basic-programmed microcomputer.     

Preparation of a permethylated β‐cyclodextrin chiral stationary phase by one‐pot hydrosilylation and immobilization at the C2 position for chiral high‐performance liquid chromatography

A novel cyclodextrin intermediate, mono‐2^A‐allylcarbamido‐2^A‐deoxy‐permethylated β‐cyclodextrin, was synthesized by reacting allylamine and newly prepared mono‐2^A‐azido‐2^A‐deoxy‐permethylated β‐cyclodextrin by the Staudinger reaction and anchored onto porous silica beads by a one‐pot hydrosilylation and immobilization procedure to afford a novel chiral stationary phase. This stationary phase acts as a new member of the previous chiral stationary phase series immobilized on the cyclodextrin C2 position. This stationary phase depicted enantiomeric separation abilities toward a series of bicyclic and tricyclic racemates under reversed‐phase conditions. The resolutions for hesperetin and naringenin achieved on the current phase reached 3.91 and 1.11, respectively, much higher than the previous permethylated β‐cyclodextrin with the linkage at the C6 position.     

Ionic liquid-modified silica as a new stationary phase for chromatographic separation

A new stationary phase based on silica modified with 1-methyl-3-propylimidazolium chloride was synthesized and characterized in this paper. A derivative of 1-methyl-3-propylimidazolium chloride was used to chemically modify the surface of silica particles to act as the stationary phase for HPLC. The modified particles were characterized by Fourier Transform Infrared (FT-IR), ¹³C NMR spectroscopy and thermogravimetric analysis (TGA). The surface modification procedure rendered particles with a surface coverage of 0.89 μmol/m² of alkylimidazolium chloride. Columns packed with the modified silica and blank silica particles were tested under HPLC conditions. Preliminary evaluation of the stationary phase for HPLC was performed using aromatic compounds as model compounds. The separation mechanism appears to involve multiple interactions including ion exchange, hydrophobic and electrostatic interactions.     

Preparation and Characterization of a Microwave-Immobilized Fluorinated Stationary Phase for RP-LC

A fluorinated stationary phase was prepared through the immobilization of poly(methyl-3,3,3-trifluoropropylsiloxane) onto 5 μm Kromasil silica by microwave irradiation. The best conditions of immobilization time and temperature were determined by central composite design and response surface methodology. Physical–chemical characterizations (IR, ²⁹Si NMR and elemental analysis) confirmed that the polymer was attached onto the chromatographic support by different mechanisms that resulted in a percent carbon loading of 10%. Some pharmaceuticals were completely separated with the fluorinated stationary phase using a simple mobile phase while the same separation was not possible with a C18 stationary phase.     

Preparation and characterization of a new HPLC C18 reversed phase containing thiocarbamate groups

A HPLC stationary phase that possesses an internal thiocarbamate functional group is described. The new C18-thiocarbamate silane was synthesized by the reaction of a trifunctional alkoxysilane with a mercaptan. The silylant agent was bonded to silica (5 μm) and the new stationary phase was then endcapped. Surface characteristics of the packing before and after chemical modification with HMDS and TMCS were determined by different physico-chemical methods, such as elemental analysis and infrared and solid-state ¹³C and ²⁹Si nuclear magnetic resonance spectroscopies. Chromatographic properties of the C18-thiocarbamate silica were evaluated under reversed phase conditions by separation of four different test mixtures that including compounds from the Engelhardt, Tanaka, and Neue test mixtures. Chromatographic evaluations of the C18-thiocarbamate phase show promising results for the separation of basic analytes.     

Synthesis and chromatographic evaluation of phenyl/tetrazole bonded stationary phase based on thiol‐epoxy ring opening reaction

A silica‐based reversed‐phase stationary phase bonding with phenyl and tetrazole groups was synthesized by thiol‐epoxy ring opening reaction. The bonded groups could not only provide hydrophobic interaction, but also π–π, hydrogen bonding, electrostatic interactions, and so on. The results of characterization with elemental analysis and solid‐state ¹³C cross‐polarization magic‐angle‐spinning NMR spectroscopy indicated the successful preparation of phenyl/tetrazole sulfoether bonded stationary phase. Chromatographic evaluation revealed that phenyl/tetrazole sulfoether bonded stationary phase behaved well under the reversed‐phase mode. The column parameters (H, S*, A, B, and C) showed different selectivity compared with some typical commercial columns, and it was validated by the separation of estrogen, ginsenoside, alkaloid samples. Based on the different selectivity between phenyl/tetrazole sulfoether bonded stationary phase and C18 columns, phenyl/tetrazole sulfoether bonded stationary phase also showed potential to construct a 2D reversed‐phase liquid chromatography system with C18. And it was verified by the separation of corydalis tuber and curcuma zedoary extracts.     

A Nano-Chip-LC/MS<Superscript><Emphasis Type="Italic">n</Emphasis></Superscript> Based Strategy for Characterization of Modified Nucleosides Using Reduced Porous Graphitic Carbon as a Stationary Phase

LC/MS analysis of ribonucleosides is traditionally performed by reverse phase chromatography on silica based C18 type stationary phases using MS compatible buffers and methanol or acetonitrile gradients. Due to the hydrophilic and polar nature of nucleosides, down-scaling C18 analytical methods to a two-column nano-flow setup is inherently difficult. We present a nano-chip LC/MS ion-trap strategy for routine characterization of RNA nucleosides in the fmol range. Nucleosides were analyzed in positive ion mode by reverse phase chromatography using a 75 μ × 150 mm, 5 μ particle porous graphitic carbon (PGC) chip with an integrated 9 mm, 160 nL trapping column. Nucleosides were separated using a formic acid/acetonitrile gradient. The method was able to separate isobaric nucleosides as well as nucleosides with isotopic overlap to allow unambiguous MS ⁿ identification on a low resolution ion-trap. Synthesis of 5-hydroxycytidine (oh⁵C) was achieved from 5-hydroxyuracil in a novel three-step enzymatic process. When operated in its native state using formic acid/acetonitrile, PGC oxidized oh⁵C to its corresponding glycols and formic acid conjugates. Reduction of the PGC stationary phase was achieved by flushing the chip with 2.5 mM oxalic acid and adding 1 mM oxalic acid to the online solvents. Analyzed under reduced chromatographic conditions oh⁵C was readily identified by its MH⁺ m/z 260 and MSⁿ fragmentation pattern. This investigation is, to our knowledge, the first instance where oxalic acid has been used as an online reducing agent for LC/MS. The method was subsequently used for complete characterization of nucleosides found in tRNAs using both PGC and C18 chips.     

The elimination of potassium activity in activation analysis by isotopic exchange on columns

The possibility of the elimination of⁴²K-activity by isotopic exchange in columns was investigated with several potassium salts as the stationary phase. A method using potassium phosphotungstate is described and examples are given.     

Silica spheres coated with C18‐modified gold nanoparticles for capillary LC and pressurized CEC separations

Nonporous monodispersed silica spheres of 1.3 μm were coated with gold nanoparticles (AuNPs) and subsequently coated with n‐octadecanethiol. By transmission electron microscopy analysis, the average diameter of the AuNPs on the silica spheres was determined to be 12 nm. The chromatographic and electrochromatographic properties of self‐assembled n‐octadecanethiol AuNP‐coated silica microspheres (C18‐AuNPs‐SiO₂) were investigated using a group of nonpolar PAHs. The stationary phase appears to display a characteristic reversed‐phase behavior. Higher separation efficiency and shorter separation times were obtained using pressurized CEC (pCEC) compared with capillary LC (CLC). A maximum column efficiency of about 2.5×10⁵ plates per meter and less than 18 min separation time for benzene were obtained in pCEC while only 66 507 plates per meter and an analysis time of nearly 100 min were observed in CLC mode. A chemical stability test of the C18‐AuNPs‐SiO₂ stationary phase under extremely high and low pH conditions demonstrated that it is stable at pH 12 and 1 for at least 60 h. The results confirm that C18‐AuNPs‐SiO₂ possesses a high rigidity to withstand high packing pressures and can be used as a good stationary phase for CLC and pCEC.     

The Development of the In Situ Modification of 1st Generation Analytical Scale Silica Monoliths

Analytical scale silica monoliths are commercially limited to three column selectivities (bare silica, C8 and C18). An in situ modification is reported in detail to overcome this barrier and allow for any functionality of choice to be bonded to the silica surface of the monolithic stationary phase support. The modification method was conducted on a commercial bare silica column to bond the C18 moiety to the silica surface through a silylation reaction. The C18 type of stationary phase was chosen, as this is the most commonly bonded functionality for the majority of stationary phases used for high-performance liquid chromatography (HPLC) separations. The C18-modified monolith’s performance was compared to a commercial C18 monolithic and a particle packed column of the same analytical scale column dimensions (100 × 4.6 mm). The modified C18 monolith proved to be of high quality with an efficiency of 73,267 N m^?1, fast analysis times (operated at flow rates up to 3 mL min^?1 using a conventional 400 bar HPLC system) and improved resolution of a set of polar and non-polar substituted aromatics in comparison to a commercial C18 monolith.     

Evaluation of the dual retention properties of stationary phases based on silica hydride: Perfluorinated bonded material

The synthesis of a new perfluorinated stationary phase based on silica hydride using a hydrosilation reaction was investigated. The material was characterized by elemental analysis, diffuse reflectance infrared Fourier transform spectroscopy and ¹³C cross‐polarization magic‐angle spinning NMR spectroscopy. The retention properties of this new material were tested in the reversed‐phase and normal‐phase mode. Variable buffer strength experiments at two pH conditions for selected polar compounds were used to compare the new phase to hydrophilic interaction liquid chromatography retention. These results and previous data reported in the literature were used to postulate differences in the retention mechanism between hydrophilic interaction liquid chromatography and silica hydride‐based stationary phases.     

Preparation, characterization and application of a stationary chromatographic phase from a new (+)-tartaric acid derivative

The preparation, characterization and application of a new stationary phase derived from 1,4-cyclohexanedione and diethyl (+)-tartrate are described. A suitable TADDOL for immobilization has been synthesized and grafted to a γ-mercaptopropylsilylated silica gel. The resulting modified stationary phase has been characterized and its ability to separate enantiomers has been studied. While the free TADDOL in solution was able to resolve a range of enantiomers, the resolving properties were lost on immobilization. Solid state ¹³C CPMAS NMR of the new stationary phase was used to explain the lack of stereoselective recognition.     

Retention and selectivity properties of carbamate pesticides on novel polar-embedded stationary phases

This study describes the use of stationary phases with polar functionality suitable for the chemical analysis of carbamates pesticides and comparing with conventional alkyl C8 and C18 phases. The emphasis of this study was to compare the selectivity and retention of the pesticides on different stationary phases, bonded onto 1.7 μm partially porous silica particles under isocratic separation condition. Four stationary phases including: phenylaminopropyl (PAP) phase, bidentate propylurea-C18 (BPUC₁₈), C8 and C18, were successfully bonded on the partially porous silica spheres as evidenced by ²⁹Si and ¹³C solid-state NMR analysis. The phenylaminopropyl phase exhibited smaller retentivity and enhanced selectivity compared to the alkyl C8 phase; the analysis time to run separation of the six carbamate pesticides (i.e., methomyl, propoxur, carbofuran, carbaryl, isoprocarb, and promecarb) on the PAP phase was threefold faster than alkyl C8 phase. In a similar manner, the BPUC₁₈ phase shows similar selectivity to that of the PAP phase, but with longer retentivity; although the BPUC₁₈ phase is characterized with a lesser degree of retentivity for the carbamate pesticides than the conventional alkyl C18 phase. We propose that π–π and weak polar interactions between the carbamate pesticides and the PAP phase dominates the separation mechanism and providing a superior selectivity; faster separation time was also achieved as a result of smaller retentivity. Whereas the C8 and C18 bonded phases exhibits only hydrophobic interactions with the pesticides, leading to larger retentivity. The BPUC₁₈ phase is shown to interact via polar–polar interactions in addition to hydrophobic interactions with the pesticides, providing similar selectivity with the PAP phase but with larger retentivity.     

Evaluation of Alkyl Bonded Silica and Solvent Phase Modifiers for the Efficient Elution of Basic Drugs on HPLC

Abstract

Nine representative drugs were used to evaluate the effects of alkyl bonded stationary phases containing type A and type B silica and the effects of an amine modifier on the efficiency of high performance liquid chromatographic elution of basic and acidic drugs. The theoretical plate count and asymmetry factor of the eluted peaks were compared to that of acetophenone as a reference to the maximal efficiency of each system evaluated. Zorbax^R C₈ was used as the stationary phase prepared from type A silica and Zorbax RX^R was used as the stationary phase prepared from the type B silica. The theoretical plate count and asymmetry factor of acetophenone was observed to be the same on both columns when analyzed in an acidic aqueous/acetonitrile mobile phase. An improvement in the efficiency and peak shape of the amine containing compounds was observed using the Zorbax RX^R stationary phase as compared to the efficiency and peak shape of these compounds on the Zorbax^R C₈ stationary phase. Interestingly, the acidic compounds salicylic acid and mefenamic acid showed better peak shapes on the Zorbax RX^R column than on the Zorbax C₈. For all drugs studied the theoretical plate count and asymmetry factor was better on both the Zorbax^R C₈ and the Zorbax RX^R stationary phases when the amine modifier triethylamine was used in the mobile phase. Except for salicylic acid, the theoretical plate count and asymmetry factor for each drug was similar on the Zorbax^R C₈ and the Zorbax RX^R columns when the amine modifier     

Synthesis and Chromatographic Evaluation of Perfluorooctyl Stationary Phase for Separation of Basic Compounds

Fluorinated stationary phases provide unique separation effect on basic compounds, due to the fluorine atoms, and pentafluorophenyl stationary phases (PFPs) are the most widely used. Considering that some fluoroalkyls have higher fluorine contents than PFPs do, it is speculated that fluoroalkyl stationary phases should have potential new applications. Herein, we synthesized a silica-based stationary phase bonding perfluoroctyl (FC8) proved by characterization through elemental analysis and solid-state ¹³C cross-polarization/magic-anglespinning nuclear magnetic resonance. The chromatographic behavior of the stationary phase was evaluated with test compounds. In addition, to further study the applicability of FC8 materials, Corydalis decumbens (Thunb.) Pers. fraction, considered as a challenging medicine on reversed-phase chromatography columns, was chosen as a test sample. Results demonstrated that the FC8 stationary phase had better and more satisfactory separation performance than the PFP stationary phase on basic compounds.

     

Liquid chromatographic separation and thermodynamic investigation of stereoisomers of darunavir on Chiralpak AD‐Hcolumn

Liquid chromatographic separation of stereoisomers of darunavir on Chiralpak AD‐H, a column containing the stationary phase coated with amylose tris(3,5‐dimethylphenylcarbamate) as a chiral selector, was studied under normal‐phase conditions at different temperatures between 20 and 50°C. The effect of quality and quantity of different polar organic modifiers viz: methanol, ethanol, 1‐propanol, and 2‐propanol in the mobile phase as well as column temperature on retention, separation, and resolution was investigated and optimized. The optimum separation was accomplished using a mobile phase composed of n‐hexane/ethanol/diethyl amine (80:20:0.1 v/v/v) at 40°C. Apparent thermodynamic parameters ΔH⁰ and ΔS* were derived from the Van't Hoff plots (lnk′ versus 1/T) and used to explain the strength of interactions between the stereoisomers and amylose tris(3,5‐dimethylphenylcarbamate) coated chiral stationary phase.