Good Peak Symmetry Research Articles

A Novel RP-HPLC Method for Simultaneous Estimation of Vilanterol Trifenatate, Umeclidinium Bromide and Fluticasone Furoate in Inhalation Dry Powder Formulation.

The dry powder inhalation formulation containing vilanterol trifenatate, umeclidinium bromide and fluticasone furoate intended for the therapy of bronchospasm related to chronic obstructive pulmonary disease and bronchial asthma was selected for the development and validation of a novel, selective, accurate, precise, quick and cost-efficient reversed-phase, high-performance liquid chromatography method. Neither an official monograph nor a single method has yet been published for the simultaneous estimation of these three compounds, which makes this method novel. The stationary phase of an ACE-C18-PFP column (250mm × 4.6mm, 5μ) was used with a mobile phase of 25-mM sodium perchlorate buffer (pH2.5 adjusted with ortho-phosphoric acid) and acetonitrile (40:60% v/v) at a flow rate of 1mL/min to optimize chromatographic variables. The column temperature was kept at 40°C, and detection was at 224nm, which was the isosbestic point of these three drugs. Well-resolved good peak symmetry was obtained for all three molecules by isocratic elution in less than 10min, and the retention times of vilanterol trifenatate, umeclidinium bromide and fluticasone furoate were found to be 3.7, 5.4 and 8.3min, respectively. The proposed method was validated as per ICH Q2 (R1) guidelines, and the calibration curves were linear in concentration ranges of 5-35μg/mL for vilanterol trifenatate, 5-80μg/mL for umeclidinium bromide and 5-150μg/mL for fluticasone furoate, with mean % recoveries of 99-100%. The limits of detection and quantitation are 0.15 and 0.45μg/mL for vilanterol trifenatate, 0.58 and 1.77μg/mL for umeclidinium bromide and 0.32 and 0.96μg/mL for fluticasone furoate, respectively. Hence, the proposed RP-HPLC technique was successfully used to quantify the inhalation formulation containing all three compounds.

Bioanalytical method development and validation of atrasentan in human plasma using verapamil as internal standard by liquid chromatography coupled with tandem mass spectrometry

A satisfactory LC-MS/MS separation and good peak symmetry for Atrasentan were obtained with Agilent, Zorbax, XDB C18 (2.1 x 50 mm ID, 5 μm), and a mobile phase containing a mixture of 5 mM Ammonium Formate buffer with 0.1% formic acid were mixed with HPLC grade Acetonitrile in the proportion of 70:30, v/v was delivered at a flow rate of 0.150 mL/min by positive ion mode (API 4000) with an injection volume of 10 µL and a run time of 3 min. Detection is performed by atmospheric pressure electrospray ionization (ESI) mass spectrometry in positive ion mode. The precursor to product ion transitions is m/z 5.11.600 to 354.04 for Atrasentan, and m/z 455.40 to 165.00 for Verapamil (Internal standard) were used for quantization. The retention time of Atrasentan and Verapamil (Internal standard) was found to be 1.68 & 0.96 min.

Bioanalytical method development and validation of atrasentan in human plasma using verapamil as internal standard by liquid chromatography coupled with tandem mass spectrometry

A satisfactory LC-MS/MS separation and good peak symmetry for Atrasentan were obtained with Agilent, Zorbax, XDB C18 (2.1 x 50 mm ID, 5 μm), and a mobile phase containing a mixture of 5 mM Ammonium Formate buffer with 0.1% formic acid were mixed with HPLC grade Acetonitrile in the proportion of 70:30, v/v was delivered at a flow rate of 0.150 mL/min by positive ion mode (API 4000) with an injection volume of 10 µL and a run time of 3 min. Detection is performed by atmospheric pressure electrospray ionization (ESI) mass spectrometry in positive ion mode. The precursor to product ion transitions is m/z 5.11.600 to 354.04 for Atrasentan, and m/z 455.40 to 165.00 for Verapamil (Internal standard) were used for quantization. The retention time of Atrasentan and Verapamil (Internal standard) was found to be 1.68 & 0.96 min.

Development and validation of a fast HPLC method for methyldopa enantiomers using superficially porous particle based macrocyclic glycopeptide stationary phase

Methyldopa is a centrally acting antihypertensive agent, whose pharmacological activity is attributed to the S-α-methyldopa enantiomer. The assessment of enantiomeric purity is an essential quality control test for chiral drugs, as the presence of certain impurities in a drug can interfere with the desired pharmacological effect. The present study was undertaken to evaluate different chiral stationary phases composed of macrocyclic glycopeptides (vancomycin, teicoplanin and aglycone teicoplanin) and the use of superficially porous particles for the efficient separation of methyldopa enantiomers. The reversed-phase and polar ionic elution modes were evaluated, with different proportions of solvents and additives. The influence of the flow rate and temperature on the analysis were also evaluated. Different stationary phases in different elution modes exhibited suitable selectivity and fast analysis. Total separation of the enantiomers were obtained with the teicoplanin and teicoplanin aglycone stationary phases. The teicoplanin aglycone column produced excellent resolution values and very fast separation analyzes. The use of the teicoplanin aglycone chiral stationary phase for the analysis of MDopa enantiomers is innovative and has not yet been explored in the literature. The best method was obtained and validated with teicoplanin aglycone in the reversed-phase mode using 20 mM ammonium acetate buffer pH 4.0/MeOH (20:80), column temperature of 45 °C and flow rate of 1.0 mL/min, which presented a total run time less than 5 min, a resolution of 5.05 and good peak symmetry. The method can be used in pharmaceutical quality control laboratories and support the stereospecific synthesis of the drug.

A fast and simple ion-pair high performance liquid chromatography method for analysis of primary bile salts in in vitro digested bean samples

Bile salts (BS) play a key role in cholesterol and lipid metabolism as well as in many other key metabolic pathways. High performance liquid chromatography (HPLC) is the most common technique used to analyze BS in diverse type of samples. However, current HPLC analysis methods used to analyze and quantify single BS in in vitro digested samples showed poor separation of a complex mixture of BS. In this article, we improved a standard method originally used for quantifying individual BS in food samples subjected to in vitro digestion. We also adapted a method previously developed for BS examination in human blood samples to the analysis of these molecules in chyme samples obtained during simulated gastrointestinal digestion. Our method was simple and achieved a fast and successful separation and quantification of four primary BS (sodium salts of taurocholic, glycocholic, taurochenodeoxycholic and glycochenodeoxycholic acids).•A method used to analyze bile salts in human blood samples has been adapted to separate and quantify four primary bile salts in in vitro digested bean samples.•Addition of an ion-pair reagent led to complete separation of glycine and taurine conjugates of chenodeoxycholic and cholic acids within 10 min, and achieved good peak symmetry.•The minimum BS concentration that could be measured was as low as 0.03125mM.

Fast and sensitive detection of acrolein in environmental water samples without derivatization using liquid chromatography tandem mass spectrometry.

A fast and sensitive SPE-LC-MS/MS method for the determination of acrolein in environmental water samples using activated charcoal as SPE adsorbent was developed. The novelty of this study consists in acrolein extraction, separation and detection without the need of a derivatization process. Physicochemical properties of acrolein, such as low molecular weight and high polarity represent real challenges for extraction, separation, and detection of this pollutant using SPE-LC-MS/MS. These were addressed by choosing a suitable chromatographic column which ensures a good peak symmetry and retention for the analyte, as well as the choice of SPE adsorbent suitable for retaining very polar compounds like acrolein from the aqueous matrix. The chromatographic column was a Synergi Fusion RP (150 × 2.0 mm, 4.0 μm) with a C18 stationary phase modified with polar embedded amide groups. Activated charcoal adsorbent used as SPE extraction media was able to extract efficiently highly polar molecules such as acrolein and 13C3-acrylamide (internal standard) from water samples. Using this method, the obtained extraction recovery for acrolein was 88% at a 50 ng/L concentration level. Overall method quantitation limit (LOQ) for acrolein in water was established at 3.8 ng/L. The newly developed SPE-LC-MS/MS method was successfully applied to detect acrolein occurrence in wastewater and drinking water samples. Acrolein level in these samples ranged from LOQ to 122 ng/L.

Development and Validation of High-Performance Liquid Chromatographic Method for Estimation of Olmesartan Medoxomil in Rat Lymph

:The present research work describes the development of a simple, rapid and sensitive liquid chromatographic method for estimation of olmesartan medoxomil (OLM) in rat lymph. Initial method development involved parameters screening to identify the critical variables and chromatographic separation was performed on a C18-column using acetonitrile and water (with 0.1% orthophosphoric acid, pH 3.5) at 40:60 ratios as mobile phase with flow rate of 1 mL.min-1 and UV detection at 243 nm. The retention time for OLM was observed at 5.8 min along with good peak symmetry, theoretical plates, absence of any tailing and interference of peak of drug with lymph fractions. Validation of the developed method indicated linearity between 100-600 ng.mL-1 along with excellent accuracy, precision and robustness with percent variation in RSD ± 2%, and good sensitivity with LOD and LOQ of 10.1 ng.mL-1 and 30.8 ng.mL-1, respectively. Further, developed method was found to be suitable for application in lymph kinetic study of nanoformulation of OLM administered to rats through peroral. Overall, the studies corroborated successful development and application of developed liquid chromatographic method of OLM in rat lymph.

Poly(4-vinylpyridine) based novel stationary phase investigated under supercritical fluid chromatography conditions

A novel poly(4-vinylpyridine) based stationary phase was investigated for its performance under supercritical fluid chromatography (SFC) mode. Due to its unique structure, this stationary phase has high molecular planarity recognition ability for aromatic samples possessing the same number of aromatic rings and π-electrons. Taking advantage of the planarity recognition ability observed, separations of structurally similar polycyclic aromatic hydrocarbons and steroids were achieved. This novel stationary phase afforded good peak symmetry for both acidic and basic active pharmaceutical ingredients even when excluding the use of additives such as acids, bases, and salts. These findings may be attributed to the polymeric pyridyl groups covalently-attached on silica gel, which will effectively shield the undesirable interaction between residual silanol groups on the surface and the analytes. Moreover, the properties of pyridyl group on the selector can be reversibly tuned to cationic pyridinium form by eluting trifluoroacetic acid containing modifier. Column robustness toward cycle durability testing was also confirmed.

Simultaneous elastase-, hyaluronidase- and collagenase-capillary electrophoresis based assay. Application to evaluate the bioactivity of the red alga Jania rubens

There have been many efforts to search for affordable and efficient cosmetic ingredients from natural sources and to evaluate their bioactivities using eco-responsible tools. Hyaluronidase, elastase and collagenase are responsible for the degradation of the main components of the extracellular matrix, namely the hyaluronic acid, elastin and collagen, respectively. The aim of this work was to develop a single capillary electrophoresis method to monitor simultaneously the activities of these three enzymes, without reactant immobilization or radioactivity use. The developed approach was used to evaluate the bioactivity of the red alga Jania rubens after microwave- or electrochemical-assisted extraction. For this purpose, the incubation time, the reactant concentrations, the separation buffer and the detection system were carefully chosen. CE with double detection system, LIF and HRMS connected in series, was used to ensure the simultaneous analysis of the substrates and products of the three enzymatic reactions. The optimized enzymatic conditions allowed the use of the same protocol to assess the 3 enzyme activities. These conditions consisted of 10 min pre-incubation of the enzyme (with alga extract) at 37 °C; 10 min incubation with the substrate at 37 °C and 10 min stop-time at 90 °C. 1.4 nL of each reaction mixture were co-injected into a 85 cm total length capillary using short-end injection. Ammonium acetate (50 mM, pH 9.0) was used for electrophoretic separation. All substrates and products were simultaneously detected in less than 10 min with good peak symmetry and efficiency, sufficient intra-day and inter-day repeatabilities (RSD < 4.5%; n = 3) and excellent LOQ (<5 nM). The results obtained using this multiple CE-based enzymatic assay showed the significant effect of Jania rubens ethanolic extracts on elastase, hyaluronidase and the metalloproteinase MMP-1.

Stress degradation of edaravone: Separation, isolation and characterization of major degradation products.

In the present study the International Conference on Harmonization-prescribed stress degradation was carried out to study the degradation profile of edaravone. To establish a Quality by Design (QbD)-assisted stability-indicating assay, the reaction solutions in which different degradation products were formed were mixed. Plackett Burman and central composite design were used to screen and optimize experimental variables to resolve edaravone and its impurities with good peak symmetry using an RP C18 column. The method was validated according to International Conference on Harmonization guidelines. Seven unknown and two known degradation products were identified and characterized by LC-MS/MS. Two major degradation products formed under thermal degradation were isolated and characterized as 4-(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl-4-(4,5-dihydro-5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-3-methyl-1-phenyl-1H-pyrazol-5(4H)-one and 3-hydroxy-dihydro-thiazolo[1-(2-methyl-buta-1,3dienyl)-1-phenylhydrazine]5-one. The degradation pathways of degradants were proposed based on m/z values.

Determination of genotoxic epoxide at trace level in drug substance by direct injection GC/MS

A novel direct injection gas chromatography method coupled with selective ion monitoring mass spectrometry (GC/SIM-MS) was developed for quantitation of trace levels of high boiling point (HBP) epoxide genotoxic impurity (GTI) in drug substance. The injector temperature was optimized with the aims to minimize matrix effects and enhance SIM signal response. The final injector temperature 160°C was selected after balancing between these two factors. The column screening was conducted as well and MN OPTIMA delta-3 silica capillary column was selected since it showed good peak symmetry without column bleeding. The good linearity was established for the concentration in the range from 0.0045μg/mL to 0.5μg/mL with a R2=0.9999. The limit of detection (LOD) and the limit of quantitation (LOQ) were 0.0014μg/mL and 0.0045μg/mL, respectively. The recovery which ranged from 95.0% to 112.5% could meet the ICH acceptance criteria. The validation results demonstrated the good linearity, precision and accuracy of the method which can be further adopted as an adequate quality control tool for quantitation of epoxide impurity at trace levels in drug substance and drug product.

3,5-Dinitrobenzoyl-9-amino-9-deoxy-9-epiquinine as Pirkle-Anion Exchange Hybrid-Type Chiral Selector in High-Performance Liquid Chromatography

A new chiral stationary phase was designed by introducing 9-amino-9-deoxy-9-epiquinine, one of the most versatile organocatalysts in asymmetric synthesis, as chiral scaffold. The derivatization of its amino group with the 3,5-dinitrobenzoyl (DNB) fragment provided hydrogen bonding and π–π donor/acceptor systems in addition to the quinoline and quinuclidine moieties having two nitrogen atoms with different basicities. The selector offers multiple interaction sites in both typical of the Pirkle-type phases and classical of weak-anion-exchanger phases. The immobilization step took place through thiol-ene addition onto 3-mercaptopropyl-silica gel and gave a grafting density of 180 µmol of chiral selector per gram of silica. A silica with reduced particle size (Daisogel silica, pore size 120 A, particle size 2.5 µm, and specific surface area 343 m2 g−1) has been employed to improve the efficiency and the speed of separations. The chiral stationary phase was packed in a small format column (50 × 4.6 mm) that allowed, by van Deemter analysis, 180,000 plates/m and approximately 5.1 µm of plate height. The ability of chiral discrimination was then studied with more than 30 test compounds using both polar-organic and normal phase conditions. In polar-organic mode, N-protected amino acids, α-aryloxy carboxylic acids, as well the non-steroidal anti-inflammatory profens were analyzed. Interesting results were obtained in normal phase elution, where the chiral selector behaves like a Pirkle-type stationary phase. Aryl amides, esterified DNB-amino acids, benzodiazepines, and binaphthol were well resolved with a very good peak symmetry and in short analysis time (mainly in less than 5 min).

Development of an analytical method for the determination of polyphenolic compounds in vegetable origin samples by liquid chromatography and pulsed amperometric detection at a glassy carbon electrode

A sensitive and accurate method for the determination of polyphenolic compounds in artichoke bract extracts and olive mill wastewaters by liquid chromatography coupled with pulsed amperometric detection at a glassy carbon working electrode was developed. Preliminary experiments were carried out by cyclic voltammetry to investigate the electrochemical behavior of polyphenols under different mobile phase compositions, and to test the detection and cleaning electrode potentials. Chromatographic separations were performed by using a core-shell C18 column, eluted with acetic acid and acetonitrile, by combined concave-linear binary gradients. Under the optimized experimental conditions, a good column efficiency and peak symmetry were observed, also for stereo and positional isomeric compounds. The developed three-step potential waveform for pulsed amperometric detection was successfully applied for the sensitive chromatographic determination of polyphenols in artichoke extracts and olive mill wastewaters. Linearity, precision and sensitivity of the proposed method have been evaluated. A wide linear range of response (up to 20mg/L) has been obtained for all the investigated compounds. Detection and quantification limits in the vegetable origin sample extracts were in the range 0.004–0.6mg/L and 0.01–2mg/L, respectively, while the injection-to-injection repeatability (n=6) ranged from 5 to 13%. The obtained results confirmed the excellent sensitivity of the electrochemical detection, and its suitability for the determination of electroactive polyphenolic compounds at low concentration levels.

Determining Urea Levels in Exhaled Breath Condensate with Minimal Preparation Steps and Classic LC-MS

Exhaled breath condensate (EBC) provides a relatively easy, non-invasive method for measuring biomarkers of inflammation and oxidative stress in the airways. However, the levels of these biomarkers in EBC are influenced, not only by their levels in lung lining fluid but also by the volume of water vapor that also condenses during EBC collection. For this reason, the use of a biomarker of dilution has been recommended. Urea has been proposed and utilized as a promising dilution biomarker due to its even distribution throughout the body and relatively low volatility. Current EBC urea analytical methods either are not sensitive enough, necessitating large volumes of EBC, or are labor intensive, requiring a derivatization step or other pretreatment. We report here a straightforward and reliable LC-MS approach that we developed that does not require derivatization or large sample volume (∼36 µL). An Acclaim mixed-mode hydrophilic interaction chromatography column was selected because it can produce good peak symmetry and efficiently separate urea from other polar and nonpolar compounds. To achieve a high recovery rate, a slow and incomplete evaporation method was used followed by a solvent-phase exchange. Among EBC samples collected from 28 children, urea levels were found to be highly variable, with a relative standard deviation of 234%, suggesting high variability in dilution of the lung lining fluid component of EBC. The limit of detection was found to be 0.036 µg/mL.

Strong Anion Determination in Biological Fluids by Capillary Electrophoresis for Clinical Diagnostics

New methods for quantitative analysis of strong anions are required for diagnostic testing of human diseases. Current techniques suffer from poor selectivity and/or long analysis times that are not amenable for labile anions in high-saline or volume-restricted samples. We introduce a rapid assay (<5 min) based on capillary electrophoresis (CE) with indirect UV detection for simultaneous analysis of sulfate, sulfite, and chloride in human urine, plasma, and sweat specimens. Remarkable selectivity for strong anions is achieved by using an acidic background electrolyte under reversed polarity that results in electrokinetic rejection of matrix interferences at the capillary inlet. A dual co-ion probe system consisting of 5 mM naphthalene disulfonate (NDS) and 5 mM naphthalene trisulfonate (NTS) in 0.4 M formic acid, pH 2.0 is developed for detection of UV transparent anions (S/N ≈ 3, 60 μM with a 25 μm inner diameter fused-silica capillary) with good peak symmetry and baseline stability. Due to the chemical reactivity of sulfite, dilute formaldehyde is used as a reagent to form an acid-stable hydroxymethylsulfonate adduct. Method validation confirmed excellent linearity (R(2) > 0.999), good accuracy (mean bias ≈7%), and acceptable long-term reproducibility (CV < 10%) over 20 days. The assay allows for artifact-free determination of sulfate and sulfite with consistent results for chloride when compared to standard electrochemical methods (R(2) > 0.975). Preliminary data suggest that kidney-stone formers have lower urinary sulfate excretion relative to non-kidney-stone patient controls (p = 0.0261). CE offers a selective yet robust platform for routine analysis of strong anions that is needed for confirmatory testing of cystic fibrosis, sulfite oxidase deficiency, urolithiasis, and other disorders of sulfur metabolism and/or anion transport.

Liquid crystal-based molecularly imprinted nanoparticles with low crosslinking for capillary electrochromatography

In this work, molecularly imprinted polymer (MIP) nanoparticles were prepared at a low level of crosslinking in the presence of liquid crystalline monomer as physical crosslinker to replace the most of chemical crosslinker. The imprinted nanoparticles against d-zopiclone were synthesized by precipitation polymerization using a mixture of methacrylic acid, ethylene glycol dimethacrylate, and liquid crystalline monomers. The resulting d-zopiclone imprinted nanoparticles were evaluated in capillary electrochromatography by partial filling technique. The resolution of enantiomer separation achieved on the d-zopiclone-imprinted nanoparticles was up to 3.29 and the column efficiency of zopiclone enantiomer (up to 66,900 plates/m) with good peak symmetry was obtained. The imprinted nanoparticles prepared in the presence of liquid crystalline monomer can retain affinity and specificity for template even when prepared with a level of cross-linker as low as 5%.

Spectroscopic and chromatographic characterisation of a pentafluorophenylpropyl silica phase end-capped in supercritical carbon dioxide as a reaction solvent

This research uses solid-state nuclear magnetic resonance (NMR) spectroscopy to characterise the nature and amount of different surface species, and chromatography to evaluate phase properties of a pentafluorophenylpropyl (PFPP) bonded silica phase prepared and end-capped using supercritical carbon dioxide (sc-CO2) as a reaction solvent. Under sc-CO2 reaction conditions (at temperature of 100°C and pressure of 414bar), a PFPP silica phase was prepared using 3-[(pentafluorophenyl)propyldimethylchlorosilane] within 1h. The bonded PFPP phase was subsequently end-capped with bis-N,O-trimethylsilylacetamide (BSA), hexamethyldisilazane (HMDS) and trimethylchlorosilane (TMCS) within 1h under the same sc-CO2 reaction conditions (100°C/4141bar). Elemental microanalysis, thermogravimetric analysis (TGA), and scanning electron microscopy (SEM) were used to provide support data to solid-state NMR and chromatographic evaluation. Results revealed a surface coverage of 2.2μmol/m2 for the non-end-capped PFPP silica phase while the PFPP phase end-capped with BSA gave a higher surface coverage (3.9μmol/m2) compared to HMDS (2.9μmol/m2) and TMCS (2.8μmol/m2). 29Si CP/MAS NMR analysis of the PFPP end-capped with BSA shows a significant decrease in the amount of Q3 (free silanols) and Q4 (siloxane groups) species, coupled with the absence of the most reactive Q2 (geminal silanols) in addition to increased amount of a single resonance peak centred at +13ppm (MH) corresponding to SiO*SiCH3 bond. 13C CP/MAS NMR shows the resonance corresponding to the propyl linkage (CH3CH2CH2) and methyl groups (Si(CH3)n) confirming successful silanisation and endcapping reactions in sc-CO2. Chromatographic evaluation of the BSA end-capped PFPP phase with Neue text mixture revealed improved chromatographic separation as evidenced in the enhanced retention of hydrophobic markers and decreased retention for basic solutes. Moreover, chromatography revealed a change in column selectivity for the BSA end-capped PFPP phase with dipropylphthalate eluting before naphthalene, indicating decreased silanol groups and increased hydrophobicity. The extend of BSA end-capping as measured by the increase in column efficiency (67,260N/m vs. 60,480N/m) on a 2.1 i.d.×50mm column, methylene group selectivity (αCH2=2.27 vs. 2.14) and decreased silanophilic interactions (S=3.7 vs. 4.10) indicate that the increase in carbon loading (3.9μmol/m2 vs. 2.2μmol/m2) and improvement in chromatography in good peak shape and symmetry is attributed to end-capping with trimethylsilyl groups.

Molecularly imprinted nanoparticles with nontailing peaks in capillary electrochromatography

The combination of microparticles of molecularly imprinted polymers (MIPs) with partial filling capillary electrochromatography (CEC) has previously been demonstrated for the enantiomer separation. In this paper, precipitation polymerization was used to prepare d-zopiclone imprinted nanoparticles (50–80nm) by a strategy of the dilution of pre-polymerization mixtures. The influence of some important parameters on the preparation of MIPs nanoparticles, including template to monomer ratio, type and amount of cross-linking monomer, and functional monomer composition ratio were investigated. In addition, the effect of separation condition, e.g., organic modifier content, pH value and salt concentration of buffer, on the electrochromatographic behavior of the MIP nanoparticles were studied. In spite of lower selectivity factor (1.11), high column performance (theoretical plates 41,400) of template was obtained and the resolution of enantiomers separation was 4.75 under the optimized conditions. Compared to the previously reported MIP microparticles, the MIP nanoparticles showed good peak symmetry and an ability of high speed separation (<15min) in CEC mode.

Electroneutral silica-based hybrid monolith for hydrophilic interaction capillary electrochromatography

A novel electroneutral and polar silica-based hybrid monolith was developed by an in situ copolymerization of 2-hydroxyethylmethacrylate (HEMA) and polyhedral oligomeric silsesquioxane methacryl substituted (POSS-MA), and successfully employed for hydrophilic interaction capillary electrochromatography (HI-CEC). A good mechanical stability of the prepared monolith was gained with the permeability decreasing from 6.52×10−14m2 to 4.61×10−14m2 when mobile phase changed from ACN to water. A significant cathodal EOF was obtained through attracting ions from the mobile phase despite the fact that it was devoid of ionizable functional groups on the surface of stationary phases, and a typical HI-CEC mechanism was achieved. The morphologies of the hybrid silica-based monolithic matrixes were observed, and the performance of this silica-based hybrid monolith was also investigated. Satisfactory column performances were carried out for both the neutral and charged analytes with HI-CEC. The analytes including uncharged amides and phenols, charged nucleic acid bases and nucleosides and enkephalins, were well separated with good peak symmetry. High separation efficiencies of charged alkaloids and enkephalins could get up to 145,000plates/m and 75,000plates/m, respectively.

Chromatography Using a Water Stationary Phase and a Carbon Dioxide Mobile Phase

A novel chromatographic separation method is introduced which employs water (saturated with CO(2)) as a stationary phase and CO(2) (saturated with water) as a mobile phase. Since water and CO(2) have little miscibility, conditions can be attained that create a stationary phase of water lining the inside of an uncoated stainless steel capillary. Because altering temperature and pressure can change both the density of the mobile phase and the polarity of the stationary phase, these experimental parameters offer good flexibility for optimizing separations and allow for different gradient programmed separation options. Further, since this method is free of organic stationary and mobile phase components, it is environmentally compatible and allows the use of universal flame ionization detection. This system offers very good sample capacity, peak symmetry, and retention time reproducibility (∼1% RSD run-to-run, ∼4% RSD day-to-day). Analytes such as alcohols, carboxylic acids, phenols, and tocopherols are employed to investigate this relatively inexpensive and robust method. As an application, the system is used to quantify ethanol in alcoholic beverages and biofuel and to analyze caffeine levels in drinks. In all cases, quantitative results are obtained with quick throughput times and often little need for sample preparation.