High-performance Liquid Chromatographic Separation Research Articles

Enantioseparation and identification for the rationalization of the environmental impact of 4 polychlorinated biphenyls.

Polychlorinated biphenyls (PCBs) are harmful and persistent organic pollutants that have long been used in industrial manufacturing. Their persistence leads to accumulation in the food chain causing potential toxic effects. As 19 out of 78 of the chiral congeners have stable atropisomers at ambient temperature, we studied some typical enantiomers: PCB45, PCB95, PCB136, and PCB149. The chiral stationary phases OD-H and OJ-H were used for separation in analytic high-performance liquid chromatography (HPLC), as well as for collection in semi-preparative HPLC. The resolution was optimized with respect to n-hexane-based mobile phases, temperature, and flow rate. All pure enantiomers were recovered from semi-preparative HPLC within 15minutes for practical purpose. Characterization of the absolute configurations were conducted with a combination of theoretical and experimental electronic circular dichroism measurements. The enantiomers of PCB45, PCB95, PCB136, and PCB149 proved to be eluted as R>S, S>R, R>S, and S>R, respectively. Molecular structures (eg, substituent groups) and properties (eg, bond lengths, bond angles, and dipole moments) were quantitatively analyzed to understand the toxicity effect of PCBs. In summary, we have developed a well-established methodology of collection and configuration identification for analogous PCB derivatives.

Preparation of silica microspheres with a broad pore size distribution and their use as the support for a coated cellulose derivative chiral stationary phase.

A templating strategy using crosslinked and functionalized polymeric beads to synthesize silica microspheres with a broad pore size distribution has been developed. The polymer/silica hybrid microspheres were prepared by utilizing the combination of a templating weak cation exchange resin, a structure-directing agent N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride, and a silica precursor tetraethyl orthosilicate. The silica microspheres were then obtained after calcinating the hybrid microspheres. The as-prepared materials were characterized by scanning electron microscopy, mercury intrusion porosimeter, and thermal gravimetric analysis. The results showed that the starting templating beads were about 5μm in diameter and the formed silica microspheres were less than 3μm with a pore size range of 10-150nm, some pores were even extended to beyond 250nm. It was demonstrated that cellulose tris(3,5-dimethylphenylcarbamate) was readily coated onto the surface of the as-synthesized silica microspheres without any additional surface pretreatment. The coated silica microspheres were uniformly dispersed even with high loading of the chiral stationary phase, which exhibited high resolution chiral separations in high-performance liquid chromatography.

Development of a precolumn derivatization HPLC method with diode-array detection for the determination of amino sugars in peat and soil humic acids.

The work is focused on the development of a high-performance liquid chromatography method with diode-array detection for the separation and quantitation of the three most abundant amino sugars; d-glucosamine, d-galactosamine, and d-mannosamine. The high-performance liquid chromatography separation was carried out by reversed-phase chromatography on Chromolith Performance RP-18e monolithic column after acid hydrolysis (5M HCl) and precolumn derivatization of samples using diethyl ethoxymethylenemalonate. Gradient elution and a mobile phase composed of ammonium formate buffer solution (10mmol/L, pH 3.60) and methanol with flow rate of 1.0mL/min were used. The monitoring wavelength was set at 280nm. The limits of detection and quantitation for analytes ranged from 0.017 to 0.122mg/L and from 0.057 to 0.407mg/L, respectively. The proposed method was successfully applied for the determination of amino sugars in samples of humic acids isolated from different soils and peat.

MOF@SiO2 core-shell composites as stationary phase in high performance liquid chromatography

Mesoporous silica spheres were functionalized with microporous metal-organic frameworks (MOFs), namely MIL-101(Fe)_NH2 (Fe3(O)(BDC_NH2)3(OH)(H2O)2, BDC = benzene-1,4–dicarboxylate) and UiO-67 (Zr6O4(OH)4(BPDC)6, BPDC = 4,4′-biphenyldicarboxylate) to produce core-shell particles for application as stationary phase in high performance liquid chromatography (HPLC) columns using a layer-by-layer method. The composites were studied for application as stationary phase in normal phase HPLC for separation of C8-isomers, dichlorobenzene isomers, styrene and ethylbenzene. Both silica/MOF composites show the highest affinity towards the ortho substituted analytes, indicating that the selectivity is dominated by the polarity of the analytes. Moreover, the UiO-67@SiO2 composite shows high separation efficiency in the separation of dimethyl phthalate and diethyl phthalate, since the number of theoretical plates per meter is very high (37570 m−1).

Layer-by-layer preparation of 3D covalent organic framework/silica composites for chromatographic separation of position isomers.

A layer-by-layer approach was developed to prepare 3D covalent organic framework COF-300/silica composites (COF-300@SiO2) with uniform morphology as the stationary phase for high performance liquid chromatography separation of position isomers with high efficiency, selectivity and precision.

Study on the Mixed-Mode HPLC Separation of Shuang-Huang-Lian and Xue‑Bi‑Jing Injections Based on an Ionic Liquid Column

Both Shuang-Huang-Lian and Xue-Bi-Jing injections are clinically proven efficient and low cost medicines. However, the studies of their chromatographic separation are limited. In this study, a column based on ionic liquid stationary phase was applied for the first time for the analysis of these two injections. For Shuang-Huang-Lian injection, U-shaped plots of retention factors vs. mobile phase constitutions were obtained. This unique kind of separation behavior allowed a flexible adjustment of retention by tuning the mobile phase constituents. Then acids group was analyzed with hydrophilic chromatography mode, while glycosides group was analyzed with reversed phase chromatography mode. Similarly, constituents in Xue-Bi-Jing injection were analyzed with mixed mode high performance liquid chromatography (HPLC). Higher, medium and lower polarity groups were analyzed with hydrophilic interaction, reversed phase and normal phase liquid chromatography modes, respectively. Results from this work provided valuable information for the separation of complicated systems such as traditional Chinese medicine injections.

Matrix solid-phase dispersion method coupled with high-performance liquid chromatography for the simultaneous quantification of selected compounds from freeze dry pomegranate juice

In this research, a novel method was developed for the matrix solid phase dispersion (MSPD) followed by high-performance liquid chromatography (HPLC) quantification of four marker constituents (vitamin C, gallic acid, rutin, and ellagic acid) in the freeze-dried pomegranate fruit juice. Various MSPD parameters like type of dispersant, sample–dispersant ratio, solvents, its volume, and time of extraction have been optimized after many trials. Furthermore, HPLC method has been developed and optimized for the analysis of all four components. The HPLC separation was achieved using a 250 × 4.6 mm column, particle size of 5 μm, C18 reverse phase column, with a mobile phase consisting of acetonitrile and 0.05% H3PO4, in gradient elution mode with a mobile phase flow rate of 1 mL/min, using ultraviolet (UV)–visible detection at 254 nm. All calibration curves showed good linear regression (r2 ≥ 0.9925) within test ranges. The extraction recoveries of the marker constituents analyzed by MSPD methods were found as ranging from 97.5% to 103.5%. From comparing the chromatograms, validation data and other parameters like time, labor, and feasibility, we found that MSPD technique was most suitable for the analysis as compared to conventional liquid–liquid extraction technique.

Core-shell microspheres with porous nanostructured shells for liquid chromatography.

The development of new stationary phases has been the key aspect for fast and efficient high-performance liquid chromatography separation with relatively low backpressure. Core-shell particles, with a solid core and porous shell, have been extensively investigated and commercially manufactured in the last decade. The excellent performance of core-shell particles columns has been recorded for a wide range of analytes, covering small and large molecules, neutral and ionic (acidic and basic), biomolecules and metabolites. In this review, we first introduce the advance and advantages of core-shell particles (or more widely known as superficially porous particles) against non-porous particles and fully porous particles. This is followed by the detailed description of various methods used to fabricate core-shell particles. We then discuss the applications of common silica core-shell particles (mostly commercially manufactured), spheres-on-sphere particles and core-shell particles with a non-silica shell. This review concludes with a summary and perspective on the development of stationary phase materials for high-performance liquid chromatography applications.

Analysis of Glutathione in Biological Samples by HPLC Involving Pre-Column Derivatization with o-Phthalaldehyde.

Glutathione (GSH) forms conjugates with polyamines in prokaryotes and eukaryotes. There is also evidence suggesting cross-talk between GSH and polyamines to regulate cellular homeostasis and function, particularly under the conditions of oxidative stress. Because of its versatile roles in cell metabolism and function, a number of high performance liquid chromatography (HPLC) methods have been developed for glutathione analysis. Here, we describe our rapid and sensitive method for the analysis of GSH and the oxidized form of glutathione (GS-SG) in animal tissues and cells by HPLC involving pre-column derivatization with o-phthalaldehyde (OPA). OPA reacts very rapidly (within 1min) with S-carboxymethyl-glutathione at room temperatures (e.g.,20-25°C) in an autosampler, and their derivatives are immediately injected into the HPLC column without any need for extraction. This method requires two simple steps (a total of 15min) before samples are loaded into the autosampler: (a) the conversion of GS-SG into GSH by 2-mercaptoethanol; and (b) the oxidation of GSH by iodoacetic acid to yield S-carboxymethyl-glutathione. The autosampler is programmed to mix S-carboxymethyl-glutathione with OPA for 1min to generate a highly fluorescent derivative for HPLC separation and detection (excitation wavelength 340nm and emission wavelength 450nm). The detection limit for GSH and GS-SG is 15pmol/ml or 375fmol/injection. The total time for chromatographic separation (including column regeneration) is 16min for each sample. Our routine HPLC technique is applicable for analyses of cysteine and cystine, as well as polyamines and GSH-polyamine conjugates in biological samples.

Synthesis of monodisperse silica microspheres and modification with diazoresin for mixed-mode ultra high performance liquid chromatography separations.

Monodisperse silica particles with average diameters of 1.9-2.9 μm were synthesized by a modified Stöber method, in which tetraethyl orthosilicate was continuously supplied to the reaction mixture containing KCl electrolyte, water, ethanol, and ammonia. The obtained silica particles were modified by self-assembly with positively charged photosensitive diazoresin on the surface. After treatment with ultraviolet light, the ionic bonding between silica and diazoresin was converted into covalent bonding through a unique photochemistry reaction of diazoresin. Depending on the chemical structure of diazoresin and mobile phase composition, the diazoresin-modified silica stationary phase showed different separation mechanisms, including reversed phase and hydrophilic interactions. Therefore, a variety of baseline separation of benzene analogues and organic acids was achieved by using the diazoresin-modified silica particles as packing materials in ultra high performance liquid chromatography. According to the π-π interactional difference between carbon rings of fullerenes and benzene rings of diazoresin, C60 and C70 were also well separated by ultra-high performance liquid chromatography. Because it has a small size, the ∼2.5 μm monodisperse diazoresin-modified silica stationary phase shows ultra-high efficiency compared with the commercial C18 -silica high-performance liquid chromatography stationary phase with average diameters of ∼5 μm.

A comparison of electrospray ionization, atmospheric pressure chemical ionization, and atmospheric pressure photoionization for the liquid chromatography/tandem mass spectrometric analysis of bisphenols. Application to bisphenols in thermal paper receipts and U.S. currency notes.

Human health concerns have arisen for common bisphenols (BPA and BPS) as well as new structural analogs. While native sulfone bisphenols and sulfone-derivatized bisphenols are amenable to electrospray ionization (ESI), alternative ionization methods such as atmospheric pressure chemical ionization (APCI) and APPI were investigated to analyze non-derivatized, non-sulfone-containing bisphenols. Ionization of bisphenols using negative ion APPI was compared to negative ion ESI and negative ion APCI for their relative abilities to produce [M-H]- precursor ions for subsequent collision-induced dissociation (CID) in a triple quadrupole mass analyzer. Multiple reaction monitoring (MRM) transitions were optimized using the most sensitive ion transitions and coupled with high-performance liquid chromatographic (HPLC) separation to detect and quantify BPA, BPB, BPF, BPZ, BPAP, BPS, and BPSIP. APPI provides a more universal sensitivity over ESI toward the classes of bisphenols studied with detection limits of 20-50 pg on-column. The LC/APPI-MS/MS method was used to examine the levels of these seven bisphenols in thermal paper receipts and in U.S. paper currency. In both receipts and currency, BPA and BPS were the dominant bisphenols found in the paper. The measurement of BPA and bisphenol analogs in thermal paper receipts and transfer of bisphenols from receipts to currency notes is of increasing importance to assess human dermal exposure routes to this class of compounds. Equivalent sensitivity between sulfone- and non-sulfone-containing bisphenols is achievable through the use of alternate ionization sources such as APCI and APPI that circumvents tedious and time-consuming derivatization procedures to render analytical sensitivity by ESI.

Construction of a hydrazone-linked chiral covalent organic framework–silica composite as the stationary phase for high performance liquid chromatography

Covalent organic frameworks (COFs), as an emerging class of crystalline porous organic polymers, have great potential for applications in chromatographic separation owning to their fascinating crystalline structures and outstanding properties. However, development of COF materials as novel stationary phases in high performance liquid chromatography (HPLC) is just in its infancy. Herein, we report the design and construction of a new hydrazone-linked chiral COF, termed BtaMth COF, from a chiral hydrazide building block (Mth) and present a one-pot synthetic method for the fabrication of BtaMth@SiO2 composite for HPLC separation of isomers. The as-synthesized BtaMth chiral COF displays good crystallinity, high porosity, as well as excellent chemical stability. Meanwhile, the fabricated HPLC column by using BtaMth@SiO2 composite as the new stationary phase exhibits high resolution performances for the separation of positional isomers including nitrotoluene and nitrochlorobenzene, as well as cis-trans isomers including beta-cypermethrin and metconazole. Additionally, some effects such as the composition of the mobile phase and column temperature for HPLC separations on the BtaMth@SiO2 packed column also have been studied in detail. The successful applications indicate the great potentials of hydrazone-linked chiral COF–silica composite as novel stationary phase for the efficient HPLC separation.

Comparative chemical fingerprinting of Oroxylum indicum and Scutellaria baicalensis using liquid chromatography and mass spectrometry

ABSTRACTIntroduction: Identification of Oroxylum indicum and Scutellaria baicalensis provides an interesting challenge in selection of biomarker compound to be used in routine analysis. Both plants have similar phytochemical profile and are rich sources of flavones and flavone glycosides. The objective of this study was to prepare the chemical fingerprinting of O. indicum bark and S. baicalensis roots using the liquid chromatography and mass spectroscopy in single chromatographic method. Materials and methods: Extracts prepared using various solvent systems (methanol, aqueous methanol, chloroform, hexane, and water) of both plants were analyzed using C18 reverse phase column with solvent system containing acetonitrile and 0.1% formic acid. Major flavonoids were identified based on mass spectra, fragmentation pattern, and UV spectra. Results: In this article, well-resolved high-performance liquid chromatographic (HPLC) separation in both plant extracts was obtained and chemical fingerprints for both plant extracts were established and flavonoids present (baicalin, oroxylin A-7-O-glucuronide, chrysin-7-O-glucuronide, baicalein, chrysin, oroxylin-A, wogonin, skullcap flavone II) were identified as possible biomarkers. Conclusion: Mass spectrometry coupled with HPLC can be a tool for fingerprinting of various natural products used in dietary supplement industry. The fingerprint developed in the article can be used for quality evaluation as well as identifying possible adulteration of extracts of both the plants.

Comparison of ultra high performance supercritical fluid chromatography, ultra high performance liquid chromatography, and gas chromatography for the separation of synthetic cathinones.

A comparison of ultra high performance supercritical fluid chromatography, ultra high performance liquid chromatography, and gas chromatography for the separation of synthetic cathinones has been conducted. Nine different mixtures of bath salts were analyzed in this study. The three different chromatographic techniques were examined using a general set of controlled synthetic cathinones as well as a variety of other synthetic cathinones that exist as positional isomers. Overall 35 different synthetic cathinones were analyzed. A variety of column types and chromatographic modes were examined for developing each separation. For the ultra high performance supercritical fluid chromatography separations, analyses were performed using a series of Torus and Trefoil columns with either ammonium formate or ammonium hydroxide as additives, and methanol, ethanol or isopropanol organic solvents as modifiers. Ultra high performance liquid chromatographic separations were performed in both reversed phase and hydrophilic interaction chromatographic modes using SPP C18 and SPP HILIC columns. Gas chromatography separations were performed using an Elite-5MS capillary column. The orthogonality of ultra high performance supercritical fluid chromatography, ultra high performance liquid chromatography, and gas chromatography was examined using principal component analysis. For the best overall separation of synthetic cathinones, the use of ultra high performance supercritical fluid chromatography in combination with gas chromatography is recommended.

Racemic and enantiopure forms of 3-ethyl-3-phenylpyrrolidin-2-one adopt very different crystal structures.

3-Ethyl-3-phenylpyrrolidin-2-one (EPP) is an experimental anticonvulsant based on the newly proposed α-substituted amide group pharmacophore. These compounds show robust activity in animal models of drug-resistant epilepsy and are thus promising for clinical development. In order to understand pharmaceutically relevant properties of such compounds, we are conducting an extensive investigation of their structures in the solid state. In this article, we report chiral high-performance liquid chromatography (HPLC) separation, determination of absolute configuration of enantiomers, and crystal structures of EPP. Preparative resolution of EPP enantiomers by chiral HPLC was accomplished on the Chiralcel OJ stationary phase in the polar-organic mode. Using a combination of electronic CD spectroscopy and anomalous dispersion of X-rays we established that the first-eluted enantiomer corresponds to (+)-(R)-EPP, while the second-eluted enantiomer corresponds to (-)-(S)-EPP. We also demonstrated that, in the crystalline state, enantiopure and racemic forms of this anticonvulsant have considerable differences in their supramolecular organization and patterns of hydrogen bonding. These stereospecific structural differences can be related to the differences in melting points and, correspondingly, solubility and bioavailability.

Screening and confirmation of steroids and nitroimidazoles in urine, blood, and food matrices: Sample preparation methods and liquid chromatography tandem mass spectrometric separations

Veterinary drugs containing synthetic anabolic steroid and nitroimidazole active agents are not allowed for their applications in livestock of the European Union (EU). This paper presents analyses of twelve selected steroids and six nitroimidazole antibiotics at low levels (1.56μg/L–4.95μg/L and 0.17μg/kg–2.14μg/kg, respectively) in body fluids and egg incurred samples. Analyses involved clean-up procedures, high performance liquid chromatography (HPLC) separation, and tandem mass spectrometric screening and confirmatory methods. Target steroids and nitroimidazoles in samples were cleaned by two independent supported liquid extraction and solid phase extraction procedures. Separation of the selected compounds was conducted on Kinetex XB C-18 HPLC column using gradient elution. The screening methods utilised supported liquid extraction that enabled fast and cost effective clean-up. The confirmatory methods were improved by extending the number of matrices and compounds, and by introducing an isotope dilution mass spectrometry for nitroimidazoles. The new methods were validated according to the recommendation of the European Union Reference Laboratories and the performance characteristics evaluated met fully the criteria. The methods were applied to incurred samples in the proficiency tests. The obtained results of Z-scores demonstrated the applicability of developed protocols of the methods to real samples. The confirmatory methods were applied to the national monitoring program and natural contamination of prednisolone could be detected in urine at low concentration in few samples.

Homochiral metal-organic frameworks based on amino acid ligands for HPLC separation of enantiomers.

Natural amino acids are well known to form coordination polymers with transition metal ions. In this study, six homochiral metal-organic frameworks constructed from Zn2+ or Co2+ ions and various enantiopure amino acid (L-tyrosine, L-histidine, L-tryptophan and L-glutamic acid), namely [Zn(L-tyr)]n (L-tyrZn), [Zn4 (btc)2 (Hbtc)(L-His)2 (H2 O)4 ]·1.5H2 O, {[Zn2 (L-trp)2 (bpe)2 (H2 O)2 ]·2H2 O·2NO3 }n , [Co2 (L-Trp)(INT)2 (H2 O)2 (ClO4 )], [Co2 (sdba)((L-Trp)2 ] and [Co(L-Glu)(H2 O)·H2 O]∞ , were synthesized according to the methods previously reported in the literature. The six homochiral MOFs were explored as the chiral stationary phases for high-performance liquid chromatographic separation of enantiomers using hexane/isopropanol or hexane/dichloromethane as mobile phase. Various types of enantiomers such as alcohols, amines, ketones, ethers, organic acids, etc. can be resolved on these homochiral MOF columns. The results revealed that the enantioseletivities of homochiral MOFs based on amino acids as chiral bridging ligands used as stationary phases are practical in HPLC.

Separation and preparation of xanthochymol and guttiferone E by high performance liquid chromatography and high speed counter-current chromatography combined with silver nitrate coordination reaction

Xanthochymol (XCM) and guttiferone E (GFE), a pair of π bond benzophenone isomers from Garcinia xanthochymus, were once reported to be difficult or impossible to separate. The present study reports the successful separation of these two isomers through high performance liquid chromatography (HPLC), as well as their effective isolation using high speed counter-current chromatography (HSCCC) based on the silver nitrate (AgNO3) coordination reaction. First, an effective HPLC separation system was developed, achieving a successful baseline separation with resolution of 2.0. Based on the partition coefficient (K) resolved by HPLC, the two-phase solvent system was determined as n-hexane, methanol and water with the uncommon volume ratio of 4:6:1. A crude extract of Garcinia xanthochymus (0.2g) was purified by normal HSCCC and refined with AgNO3-HSCCC. Monomers of XCM and GFE were identified by HPLC, mass spectrometry (MS) and nuclear magnetic resonance (NMR). The results demonstrate the separation and isolation of π bond benzophenone isomers using ordinary octadecyl silane (C18) columns and HSCCC.

Molecular Weight Distribution of Living Chains in Polystyrene Prepared by Atom Transfer Radical Polymerization.

Living and dead chains of a polystyrene synthesized by atom transfer radical polymerization were separated and characterized by high performance liquid chromatography (HPLC), size exclusion chromatography (SEC), NMR, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The bromine end group in the living chain was quantitatively converted to a hydroxyl end group via first azidation and subsequent copper-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction with propargyl alcohol. The living chains bearing a polar end group are fully resolved from the unmodified dead chains by HPLC separation using a bare silica stationary phase. Molecular weight distributions (MWD) of the living and dead chain are characterized by SEC and MALDI-MS. The MWD of the living chains is close to a Poisson distribution. Interestingly, the elution peak of the living chains in the HPLC separation split into two. The peak split is attributed to the diastereomeric structure of the chain end by NMR and MALDI-MS analyses.

High performance liquid chromatographic separation of 228,229Th and 232,233U and their estimation by α- and γ-ray spectrometry

The activities of 228,229Th and 232,233U from an irradiated ThO2 sample were radiochemicaly separated by using high performance liquid chromatography. Plancheted sources of the separated samples were made and the amount of 232,233U and 228,229Th were estimated by using alpha and gamma-ray spectrometric techniques. These estimations are important for the Th–U fuel reprocessing cycle of advanced heavy water reactor and accelerator driven sub-critical system.