Stationary Phase For High-performance Liquid Chromatography Research Articles

Biotin-functionalized poly(ethylene terephthalate) capillary-channeled polymer fibers as HPLC stationary phase for affinity chromatography.

Native poly(ethylene terephthalate) (PET) capillary-channeled polymer (C-CP) fibers have been used as the stationary phase for high-performance liquid chromatography (HPLC) of proteins via reversed-phase and ion-exchange processes. Functionalization can be used to bring about greater selectivity through surface modification. PET fibers were treated with ethylenediamine to generate primary amine groups on the fiber surface, enabling subsequent covalent attachment of ligands. The ninhydrin test for primary amines revealed surface densities of 13.9-60.0 μmol m(-2) for PET fibers exposed for periods of 3-12 min. Here, 8-amino-3,6-dioxaoctanoic acid was linked to the EDA-treated PET fiber surface as a hydrophilic spacer, and then D-biotin was attached on the end of the spacer as an affinity ligand. The streptavidin binding capacity and binding homogeneity were studied on the biotin-functionalized PET C-CP fiber microbore column. The selectivity of the biotin surface functionalization was assessed by spiking lysate with Texas Red-labeled streptavidin and enhanced green fluorescent protein. Greater than 99% selectivity was realized. This ligand-coupling strategy from standard solid-phase peptide synthesis used in stationary phase functionalization creates great potential for PET C-CP fiber-packed HPLC columns to perform a variety of chromatographic separations.

C₁₈-bound porous silica monolith particles as a low-cost high-performance liquid chromatography stationary phase with an excellent chromatographic performance.

Ground porous silica monolith particles with an average particle size of 2.34 μm and large pores (363 Å) exhibiting excellent chromatographic performance have been synthesized on a relatively large scale by a sophisticated sol-gel procedure. The particle size distribution was rather broad, and the d(0.1)/d(0.9) ratio was 0.14. The resultant silica monolith particles were chemically modified with chlorodimethyloctadecylsilane and end-capped with a mixture of hexamethyldisilazane and chlorotrimethylsilane. Very good separation efficiency (185,000/m) and chromatographic resolution were achieved when the C18 -bound phase was evaluated for a test mixture of five benzene derivatives after packing in a stainless-steel column (1.0 mm × 150 mm). The optimized elution conditions were found to be 70:30 v/v acetonitrile/water with 0.1% trifluoroacetic acid at a flow rate of 25 μL/min. The column was also evaluated for fast analysis at a flow rate of 100 μL/min, and all the five analytes were eluted within 3.5 min with reasonable efficiency (ca. 60,000/m) and resolution. The strategy of using particles with reduced particle size and large pores (363 Å) combined with C18 modification in addition to partial-monolithic architecture has resulted in a useful stationary phase (C18 -bound silica monolith particles) of low production cost showing excellent chromatographic performance.

Quantitation of enantiomers of r-7,t-8,9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]-pyrene in human urine: evidence supporting metabolic activation of benzo[a]pyrene via the bay region diol epoxide.

Benzo[a]pyrene (BaP), a potent polycyclic aromatic hydrocarbon carcinogen, is widely distributed in the human environment. All humans are exposed to BaP through the diet and contact with the general environment; cigarette smokers have higher exposure. An important pathway of BaP metabolism proceeds through formation of diol epoxides including the 'bay region diol epoxide' 7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [BaP-(7R,8S)-diol-(9S,10R)-epoxide] and the 'reverse diol epoxide' 9S,10R-dihydroxy-7R,8S-epoxy-7,8,9,10-tetrahydrobenzo [a]pyrene [BaP-(9S,10R)-diol-(7R,8S)-epoxide]. The bay region diol epoxide is considered a major ultimate carcinogen of BaP based on studies in cell culture and laboratory animals, but the available data in humans are less convincing. The bay region diol epoxide and the reverse diol epoxide react with H2O to produce enantiomeric BaP-tetraols that are excreted in the urine. We used chiral stationary-phase high-performance liquid chromatography and gas chromatography-negative ion chemical ionisation-tandem mass spectrometry to quantify these enantiomeric BaP-tetraols in the urine of 25 smokers and 25 non-smokers. The results demonstrated that the BaP-tetraol enantiomer representing the carcinogenic bay region diol epoxide pathway accounted for 68±6% (range 56-81%) of total BaP-tetraol in smokers and 64±6% (range 46-78%) in non-smokers. Levels of the major BaP-tetraol enantiomer decreased by 75% in smokers who quit smoking. These data provide convincing evidence in support of the bay region diol epoxide mechanism of BaP carcinogenesis in humans.

Quantitative analysis of molecular interaction potentials of ionic liquid anions using multi-functionalized stationary phases in HPLC.

The molecular interaction potentials, including S (dipolarity/polarizability), A (hydrogen bonding acidity), and B (hydrogen bonding basicity), of anions are experimentally determined using multi-functionalized stationary phases in high-performance liquid chromatography (HPLC) systems. We employ three different multi-functionalized stationary phase columns (Obelisc R, Obelisc N, and Acclaim Trinity-P1) combined with two ingredients, namely, acetonitrile (ACN) and methanol (MeOH). These conditions can cause neutral, cationic, and anionic compounds to be retained. By using the retention characteristics of calibration compounds, including cations, anions, and neutral compounds, system parameters including the ionic interaction terms (zc Zc , za Za ) are evaluated using multiple linear regression, resulting in a standard deviation (SD) of 0.090-0.158 log units. Based on the system parameters and retention characteristics of the anions of interest, their molecular interaction potentials are characterized on the same scale for neutral and cationic molecules. Furthermore, to verify the determined molecular interaction potentials, we predict anion hydrophobicity. The results show that the determined S, A, and B, together with the computable descriptors E (excess molar refraction) and V (McGowan volume), can predict anion hydrophobicity with R(2) =0.982 and SD=0.167 (dimensionless).

Determination of the pore size distribution of high-performance liquid chromatography stationary phases via inverse size exclusion chromatography

Stationary phases in liquid chromatography exhibit quite different pore structures. Whereas most of the fully porous packing materials possess a narrow pore size distribution, core–shell particles are usually of rather wide pore size distribution. Recently a novel theory of size exclusion chromatography was introduced to model the effect of pore size distribution. The molecular theory of chromatography allows taking into account the kinetics of the pore ingress and egress processes, the heterogeneity of the pore sizes and polymer polydispersity as well. The novel model was applied to inverse size exclusion chromatography data. In this study, we have determined the actual pore size distribution of a number of HPLC stationary phases. Our results agree well with the results obtained with the model introduced by Knox and Scott.

An artificial receptor fabricated by target recognition determinant imprinting for selective capture of α-amanitin

This is the first reported work of artificial α-amanitin receptors which are prepared using the design and synthesis of a template molecule based on an α-amanitin recognition determinant imprinting strategy. The resultant molecularly imprinted polymers (MIPs) are evaluated using high performance liquid chromatography (HPLC), binding experiment, nitrogen adsorption measurement, and solid-phase extraction. Experiment clearly demonstrates that the MIPs as the HPLC stationary phase can specifically recognize α-amanitin from analogues. The MIPs also successfully adsorb trace amounts of α-amanitin in spiked serum samples selectively pretreated and enriched through molecularly imprinted solid phase extraction. The limit of detection and recovery is 3.0ngmL−1 and 88.5–95.9%, respectively, for α-amanitin in serum samples. The high specific adsorption and excellent selectivity of the MIPs arises from imprinting effects related to the imprinting cavity of the polymeric matrix, the metal-coordination bond, and the hydrogen bond between the receptor and ligand.

Preparation and characterization of mesoporous silicas modified with chiral selectors as stationary phase for high-performance liquid chromatography

New hybrid materials were prepared as novel chiral stationary phases (CSPs) for high-performance liquid chromatography (HPLC). Pure mesoporous silica (SM) and ethylene-bridged periodic mesostructured organosilica (PMO) were functionalized, by a post-synthesis method, with derivates of erythromycin and vancomycin. N2 adsorption–desorption measurements, XRD, FT-IR, MAS NMR, SEM, TEM and elemental analysis were used to characterize the physico-chemical properties of these mesostructured materials, before and after the modification process. The synthesized particles had non-symmetrical 3-D wormhole-like mesostructure, spherical morphology, and a mean pore diameter between 53 and 59Å. CSPs prepared were tested for the separation of four chiral β-blockers (atenolol, metoprolol, pindolol and propranolol) in normal phase (NP) and polar organic phase (PO) elution modes. Much stronger chiral interaction was observed in vancomycin-modified silicas. Results obtained in these preliminary studies will permit in future works to improve the synthesis route in order to design mesoporous materials with better performance as a chiral stationary phase for HPLC.

Fabrication of ZIF‐8@SiO2 Core–Shell Microspheres as the Stationary Phase for High‐Performance Liquid Chromatography

The unique features of high porosity, shape selectivity, and multiple active sites make metal-organic frameworks (MOFs) promising as novel stationary phases for high-performance liquid chromatography (HPLC). However, the wide particle size distribution and irregular shape of conventional MOFs lead to lower column efficiency of such MOF-packed columns. Herein, the fabrication of monodisperse MOF@SiO2 core-shell microspheres as the stationary phase for HPLC to overcome the above-mentioned problems is reported. Zeolitic imidazolate framework 8 (ZIF-8) was used as an example of MOFs due to its permanent porosity, uniform pore size, and exceptional chemical stability. Unique carboxyl-modified silica spheres were used as the support to grow the ZIF-8 shell. The fabricated monodisperse ZIF-8@SiO2 packed columns (5 cm long × 4.6 mm i.d.) show high column efficiency (23,000 plates m(-1) for bisphenol A) for the HPLC separation of endocrine-disrupting chemicals (bisphenol A, β-estradiol, and p-(tert-octyl)phenol) and pesticides (thiamethoxam, hexaflumuron, chlorantraniliprole, and pymetrozine) within 7 min with good relative standard deviations for 11 replicate separations of the analytes (0.01-0.39, 0.65-1.7, 0.70-1.3, and 0.17-0.91% for retention time, peak area, peak height, and half peak width, respectively). The ZIF-8@SiO2 microspheres combine the advantages of the good column packing properties of the uniform monodisperse silica microspheres and the separation ability of the ZIF-8 crystals.

Preparation and retention mechanism study of graphene and graphene oxide bonded silica microspheres as stationary phases for high performance liquid chromatography

Graphene oxide (GO) bonded stationary phase for high performance liquid chromatography (HPLC) was fabricated by coating GO sheets onto aminosilica microspheres via covalent coupling. Graphene (G) functionalized HPLC stationary phase was then prepared through hydrazine reduction of GO bonded silica (GO@SiO2) composite, which was the first example of using graphene as stationary-phase component for HPLC. Effective separations of the tested neutral and polar compounds on both GO@SiO2 and graphene bonded silica (G@SiO2) columns were achieved under the optimal experimental conditions. Compared with commercial C18 column, the different chromatographic performances of GO and graphene bonded columns were ascribed to their unique retention mechanisms. The polyaromatic scaffold of GO and graphene gives π–π stacking property and hydrophobic effect, and other retention mechanisms, such as π–π electron-donor–acceptor (EDA) interaction for the separation of nitroaromatic compounds and hydrogen bonding for hydroxyl and amino compounds, may also be taken into consideration. Experimental results indicated that the mixed-mode retention mechanism can facilitate the separation of analytes with similar hydrophobicity, which is a unique property compared with C18 column. Additionally, G@SiO2 showed higher affinity to aromatic analytes in contrast with GO@SiO2 and its retention mechanism was not consistent with the typical reversed phase behavior. The separation of aromatic compounds on G@SiO2 column relies primarily on the π–π stacking interaction and then the hydrophobicity, while the two interactions have equal shares on GO@SiO2 column.

Effect of Chromatographic Conditions on Enantioseparation of Bovine Serum Albumin Chiral Stationary Phase in HPLC and Thermodynamic Studies

Twelve chiral compounds were enantiomerically resolved on bovine serum albumin chiral stationary phase (BSA-CSP) by high-performance liquid chromatography (HPLC) in reversed-phase modes. Chromatographic conditions such as mobile phase pH, the percentage of organic modifier, and concentration of analyte were optimized for separation of enantiomers. For N-(2, 4-dinitrophenyl)-serine (DNP-ser), the retention factors (k) greatly increase from 0.81 to 6.23 as the pH decreasing from 7.21 to 5.14, and the resolution factor (R(s)) exhibited a similar increasing trend (from 0 to 1.34). More interestingly, the retention factors for N-(2, 4-dinitrophenyl)-proline (DNP-pro) decrease along with increasing 1-propanol in mobile phase (3%, 5%, 7% and 9% by volume), whereas the resolution factor shows an upward trend (from 0.96 to 2.04). Moreover, chiral recognition mechanisms for chiral analytes were further investigated through thermodynamic methods.

Application of resorcinarene derivatives in chemical separations

The applications of resorcinarene derivatives in modern separation techniques including high performance liquid chromatography (HPLC), gas chromatography (GC), capillary electrophoresis (CE), ion chromatrography (IC), and liquid membranes are reviewed in this paper. Resorcinarenes are macrocyclic compounds which can be modified with various substituents on upper and lower rims to provide specific functionality and selectivity. The derivatives can be adsorbed or covalently bound to the stationary phase of HPLC, GC, and IC, or used as pseudo-stationary phase in CE to separate organic or inorganic species. They have been tested as carriers to selectively transfer species from source phase to receiving phase in bulk liquid membranes, supported liquid membranes, and polymer inclusion membranes. These broad applications of resorcinarene-based macrocyclic ligands indicate that they are a promising class of compounds that are worthwhile to be explored in both synthesis and separation applications.

Influence of stereoregularity and linkage groups on chiral recognition of poly(phenylacetylene) derivatives bearing L‐leucine ethyl ester pendants as chiral stationary phases for HPLC

AbstractStereoregular poly(phenylacetylene) derivatives bearing L‐leucine ethyl ester pendants, poly‐1 and poly‐2a, were, respectively, synthesized by the polymerization of N‐(4‐ethynylphenylcarbamoyl)‐L‐leucine ethyl ester (1) and N‐(4‐ethynylphenyl‐carbonyl)‐L‐leucine ethyl ester (2) using Rh(nbd)BPh4 as a catalyst, while stereoirregular poly‐2b was synthesized by solid‐state thermal polymerization of 2. Their chiral recognition abilities for nine racemates were evaluated as chiral stationary phases (CSPs) for high‐performance liquid chromatography (HPLC) after coating them on silica gel. Both poly‐1 and poly‐2a with a helical conformation showed their characteristic recognition depending on coating solvents and the linkage groups between poly(phenylacetylene) and L‐leucine ethyl ester pendants. Poly‐2a with a shorter amide linkage showed higher chiral recognition than poly‐1 with a longer urea linkage. Coating solvents played an important role in the chiral recognition of both poly‐1 and poly‐2a due to the different conformation of the polymer main chains induced by the solvents. A few racemates were effectively resolved on the poly‐2a coated with a MeOH/CHCl3 (3/7, v/v) mixture. The separation factors for these racemates were comparable to those obtained on the very popular CSPs derived from polysaccharide phenylcarbamates. Stereoirregular poly‐2b exhibited much lower chiral recognition than the corresponding stereoregular, helical poly‐2a, suggesting that the regular structure of poly(phenylacetylene) main chains is essential to attain high chiral recognition. © 2013 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013

Preparation of a novel skeleton weak anion-exchange monolith by single electron transfer-living radical polymerization for protein separation in high performance liquid chromatography

A novel skeleton weak anion-exchange monolith was prepared by single electron transfer-living radical polymerization (SET-LRP) for high performance liquid chromatography (HPLC). In this study, vinyl ester resin was used as the monomer, ethyleneglycol dimethacrylate as the cross linking agent, carbon tetrachloride as the initiator, ferrous powder as the catalyst, and N,N,N′,N′-tetramethylethylenediamine as the ligand to supply amine-groups. The polymerization conditions were optimized. The characters of the monolith were investigated by scanning electron microscopy, Fourier transform infrared spectroscopy and mercury porosimeter, respectively. Good skeleton structure properties were obtained. Moreover, the prepared monolith was used as HPLC stationary phase to separate immune globulin G (IgG) from human plasma with high efficiency. The influences of buffer concentration and pH of the mobile phase on the separation of IgG were investigated. In addition, the monolith was also used to separate the mixture of beef serum albumin (BSA), IgG and lysozyme (Lys) successfully.

Benzimidazole modified silica as a novel reversed-phase and anion-exchange mixed-mode stationary phase for HPLC

A novel stationary phase based on benzimidazole modified silica was prepared and evaluated as reversed-phase and anion-exchange mixed-mode stationary phase for high performance liquid chromatography (HPLC). Using the phase, polycyclic aromatic hydrocarbons, phthalates, anilines and phenols were respectively separated depending on formation of different types of chemical bonds like hydrogen bonding, ππ stacking, electrostatic forces and hydrophobic interactions by reversed-phase chromatography; inorganic and organic anions were also separated mainly through anion-exchange and ππ interactions by anion-exchange chromatography with high resolution and column efficiency. The proposed benzimidazole modified silica is a promising mixed-mode stationary phase for the separation of complex samples in HPLC.

Recent Developments in High-Performance Liquid Chromatography Stationary Phases

Recent Developments in High-Performance Liquid Chromatography Stationary Phases

Study of the Molecularly Imprinted Polymers for Selective Binding of the Mono-Substituted Sulfonylurea Herbicides

A new molecularly imprinted polymer (MIP) was synthesized for the selective extraction of mono-substituted sulfonylurea herbicides, with monosulfuron as the template and acrylamide as the functional monomer. The recognition property and affinity of the MIP for monosulfuron and its analog, monosulfuron-ester, were evaluated by equilibrium adsorption and a chromatographic study. Computer modeling, including simulated annealing and semi-empirical quantum calculation, was employed to study the recognition mechanism. The computer modeling demonstrated that monosulfuron can form multiple hydrogen bonds with methacrylic acid and acrylamide, whereas monosulfuron-ester cannot form a stable complex with these two functional monomers, which aligns with the results of the rebinding experiment. The selectivity study further demonstrated that binding sites in the MIP interact with the hydrogen in the acylamino group of mono-substituted sulfonylurea. A comparison experiment also showed that monosulfuron-imprinted MIP offers better selectivity for monosulfuron-ester than the commercial C18 high-performance liquid chromatography stationary phase material.

Frozen polymerization for aligned porous structures with enhanced mechanical stability, conductivity, and as stationary phase for HPLC

A directional freezing and frozen polymerization method is developed to prepare crosslinked aligned porous polymers with improved mechanical stability. Monomer solutions are directionally frozen in liquid nitrogen to orientate the growth of solvent crystals. The frozen samples are polymerized by UV irradiation. The solvent is removed under vacuum at room temperature to produce aligned porous structure. The mechanical stability is improved by two orders of magnitude compared to the usually freeze-dried porous materials. The materials are modified with graphene and a conducting polymer to achieve conductivity at 1.9 × 10−4 S cm−1 and 5.2 × 10−6 S cm−1, with the stable aligned pore structures maintained. The aligned porous monolith is also assessed by high performance liquid chromatography (HPLC), showing fast separation of hydrocarbon compounds with low back pressure at 59 bar.

Preparation, characterization and application of a new 25,27-bis-[2-(5-methylthiadiazole)thioethoxyl]-26,28-dihydroxy-para-tert-butyl calix[4]arene stationary phase for HPLC.

A new para-tert-butylcalix[4]arene column containing thiadiazole functional groups was prepared and used for the separation of polycyclic aromatic hydrocarbons, phenolic compounds, aromatic amines, benzoic acid and its derivatives by high-performance liquid chromatography (HPLC). The effect of organic modifier content in the mobile phase on retention and selectivity of these compounds were investigated. The results indicate that the stationary phase behaves like reversed-phase packing. However, hydrogen bonding, π-π and inclusion interactions seem to be involved in the separation process. The column has been successfully employed for the analysis of clenbuterol in pork and pig casing; the limit of detection and the limit of quantitation for this method by HPLC-UV detection was 0.03 and 0.097 μg/mL, respectively; the method is demonstrated to be suitable and a competitive alternative analytical method for the determination of clenbuterol.

Enantioseparation on poly(phenyl isocyanide)s with macromolecular helicity memory as chiral stationary phases for HPLC

A series of novel optically active poly(phenyl isocyanide)s bearing achiral benzanilide pendant groups was prepared based on the noncovalent “helicity induction and memory strategy”. The polymers that exhibited an optical activity due to the macromolecular helicity memory were immobilized to 3-aminopropyl-silanized silica gel through chemical bonding or coated on the silica gel to obtain chiral packing materials for high-performance liquid chromatography (HPLC). The chiral recognition abilities toward a variety of racemic compounds with different functionalities were investigated by HPLC. The obtained CSPs separated various racemates into enantiomers, in particular, a right-handed helical poly(phenyl isocyanide) carrying achiral anilide pendants showed an excellent chiral recognition ability and completely or partially resolved nine racemates, including cyclic ether, amine, ketones and metal acetylacetonate complexes, among twelve racemates.

A new 14-membered tetraazamacrocycle-bonded silica stationary phase for reversed-phase high-performance liquid chromatography

A new high-performance liquid chromatography stationary phase has been prepared by covalently bonding 14-membered tetraazamacrocycle to silica gel using γ-chloropropyltrimethoxylsilane as coupling agent. The structure of the new material was characterized by infrared spectroscopy and elemental analysis. With 32 solutes including aromatic and aliphatic compounds, the linear solvation energy relationship method was successfully used to chromatographically evaluate the new phase in reversed phase mode. The retention property of the new phase shows evident similarity with that of ODS stationary phase, as well as distinctive, unique retention characteristics. The separations of n-alkylbenzene, carbamate and organophosphorus pesticides with diversified functional groups as well as phenolic compounds demonstrate that in addition to hydrophobic interaction, dipole–dipole interaction and hydrogen bonding interaction plus acid–base equilibrium could also be simultaneously offered by this new stationary phase, as a result excellent chromatographic performances are guaranteed.