Light-scattering detection of phospholipids resolved by HPLC

An improved method for the analysis of phospholipids by normal-phase HPLC is described. Addition of methanol and acetonitrile to a gradient based on 2-propanol/hexane/water promoted a rapid separation of major classes of bovine surfactant phospholipids (PL) by using a conventional silica column. The use of an ELSD permitted an accurate analysis of a mixture of PL. Calibration curves were linear within the range of 5–40 μg with detection limits below 1 μg for PE and PC, and CV ranged from 0.6 to 9.6%. PL present in surfactant homogenates were separated by a solid-phase extraction (SPE) procedure before HPLC analysis. This methodology gave a recovery of 95% and combined SPE-HPLC and quantification of biological PL within a 30-min run. The use of ELSD detection of the eluted compounds was precise, linear, and sensitive.     

Under‐Oil Superhydrophilic Poly(vinyl alcohol)/Silica Hybrid Nanofibrous Aerogel for Gravity‐Driven Separation of Surfactant‐Stabilized Water‐in‐Oil Emulsions

For efficient and green separation of surfactant‐stabilized water‐in‐oil (W/O) emulsions, under‐oil superhydrophilic poly(vinyl alcohol) (PVA)/silica hybrid nanofibrous aerogel is fabricated by freeze‐drying the dispersion of shortened PVA/tetraethyl orthosilicate composite electrospun nanofibers in t‐butanol, followed by heat‐treatment. Its hierarchical porous structure, observed by scanning electron microscope, consists of major and minor pores with an average diameter of 15.9 and 1.0 µm, respectively. The silica‐based crosslinking structure inside the nanofibers and the chemical linkage between them, evidenced by infrared spectroscopy, endows the nanofibrous aerogel with desirable stability in water and compression recoverability. When it is used for gravity‐driven separation of Span80 stabilized water‐in‐n‐hexane emulsion, the flux is 2083 L m^?2 h^?1 and the purity of the separated n‐hexane reaches 99.997%, corresponding to the separation efficiency of 99.79%. The nanofibrous aerogel after use is readily recycled by rinsing and freeze‐drying, without using any organic solvent, as it possesses under‐oil superhydrophilicity and prominent oil antifouling property. Differing from the previously reported separation materials, PVA/silica hybrid nanofibrous aerogel simultaneously acts as gravity‐driven filtration material and adsorption material to both absorb their coalesced water droplets and allow the separated oil to penetrate in the separation process.     

Development of a pre‐purification process for homoharringtonine

A novel pre‐purification method was developed for producing homoharringtonine from Cephalotaxus koreana, giving high purity and yield. The simple, efficient procedure involved biomass extraction, liquid–liquid extraction, and synthetic adsorbent treatment, followed by low‐pressure chromatography. The use of active clay treatment and silica gel low‐pressure chromatography in the pre‐purification process allowed for the rapid, efficient separation of homoharringtonine from interfering compounds and, compared with alternative processes, increased the yield and purity of crude homoharringtonine for subsequent high‐performance liquid chromatography (HPLC) purification. Homoharringtonine of over 52% purity could be obtained simply with high yield from biomass using this pre‐purification method, while minimizing solvent use and the scale and complexity of HPLC operations for homoharringtonine purification. Copyright © 2005 Society of Chemical Industry     

Separation and analysis of phospholipids in different foods with a light-scattering detector

A method for the separation of phospholipids (PL) from total lipids by solid-phase extraction (SPE) with reversephase C₈ cartridges is described. The method was validated with a standard mixture of PL and applied to natural food matrixes, such as egg, chicken meat, salami, and ripened cheese. The recovery of PL ranged between 93 and 99.7% and was evaluated by an organic phosphorus spectrophotometric determination. The egg powder PL fraction obtained by SPE contained about 20% (w/w) nonpolar PL material when 100–150 mg of lipids were loaded onto the cartridge. Higher percentages of nonphospholipid components (30–43%) were obtained when the amount of lipids loaded was below or above the 100–150 mg range. The purified PL fractions were analyzed by high-performance liquid chromatography (HPLC) with an evaporative light-scattering detector. Good HPLC performance was observed even with low-purity SPE fractions (43% nonphospholipid material).     

Morphology and properties of poly[2‐methoxy‐5‐(2′‐ethyl‐hexyloxy)‐para‐phenylene vinylene]/silica nanoparticle hybrid films

Poly[2‐methoxy‐5‐(2′‐ethyl‐hexyloxy)‐para‐phenylene vinylene] (MEH‐PPV)/silica nanoparticle hybrid films were prepared and characterised. Three kinds of materials were compared: parent MEH‐PPV, MEH‐PPV/silica (hybrid A films), and MEH‐PPV/coupling agent MSMA/silica (hybrid B films), in which MSMA is 3‐(trimethoxysilyl) propyl methacrylate. It was found that the hybrid B films could significantly prevent macrophase separation, as evidenced by scanning electron and fluorescence microscopy. Furthermore, the thermal characteristics of the hybrid films were largely improved in comparison with the parent MEH‐PPV. The UV‐visible absorption spectra suggested that the incorporation of MSMA‐modified silica into MEH‐PPV could confine the polymer chain between nanoparticles and thus increase the conjugation length. The photoluminescence (PL) studies also indicated enhancement of the PL intensity and quantum efficiency by incorporating just 2 wt% of MSMA‐modified silica into MEH‐PPV. However, hybrid A films did not show such enhancement of optoelectronic properties as the hybrid B films. The present study suggests the importance of the interface between the luminescent organic polymers and the inorganic silica on morphology and optoelectronic properties. Copyright © 2004 Society of Chemical Industry     

Separation and Detection of Plasmalogen in Marine Invertebrates by High‐Performance Liquid Chromatography with Evaporative Light‐Scattering Detection

We have developed a new method for determining ethanolamine plasmalogen contents in marine invertebrates. This quantification method involves derivatization of ethanolamine glycerophospholipid (EtnGpl) subclasses, alkenylacyl (plasmalogen), diacyl, and alkylacyl subclasses, by enzyme treatment and acetylation, followed by separation and detection by high‐performance liquid chromatography (HPLC) with evaporative light‐scattering detection (ELSD). This method enabled complete separation of the subclasses, and the limit of detection for plasmalogen was 200 ng (260 pmol). The peak area of plasmalogen by ELSD was unaffected by the degree of unsaturated fatty acids in EtnGpl, in contrast to ultraviolet (UV) detection. Thus, this method enables accurate determination of plasmalogen contents in various species containing marine products possessing abundant polyunsaturated fatty acids (PUFA). The method developed here was applied to marine invertebrates available in Japan. The examined marine invertebrates showed a wide range of plasmalogen contents ranging from 19 to 504 μmol/100 g wet wt. The plasmalogen levels in samples except those of class Cephalopoda and Crustacea were more than 60 mol% of EtnGpl.     

Quantification of Phorbol Esters in Jatropha curcas by HPLC‐UV and HPLC‐ToF‐MS with Standard Addition Method

Established analytic methods for the quantification of phorbol esters (PE), which are some toxic components in Jatropha curcas L., include HPLC with UV‐detection with the commercially available phorbol myristate acetate (PMA) as internal standard or HPLC coupled with MS detection with an external calibration, mostly also with PMA. The differences in the fatty acid side chains and connection to the base structure of PMA compared to PE leads to different UV absorption and MS ionization effects and cause problems for exact quantitative measurements. In this paper, a method is presented which combines both detection types and shows differences between both results. For this purpose, an extraction routine is performed on a PE‐containing seed oil to get a PE standard in high purity, which was used for a standard addition method on two real J. curcas oil samples, derived from Ghana and Mexico. Furthermore, a detection window of ±10 ppm for the high accurate ToF‐MS detection is set to eliminate isobaric interferences from co‐eluting material. Method evaluation of inter‐ and intra‐day variance as well as the recovery rate are performed and determined. With this method a limit of detection of 62 ng mL^?1 (UV) and 11 ng mL^?1 (MS) can be achieved. Practical Applications: Due to the good biological and technical properties of Jatropha curcas L., its seed oil seems perfect for the application as biodiesel feedstock. The toxicity on the other hand could cause problems when converting side products from the oil production to products of higher value. With the here described method an accurate and precise analysis procedure for the quantification of the toxic compounds namely, phorbol esters, could be applied for toxicity studies or routine checks in industry which is converting plant material from J. curcas, so that no toxic material is used for example as animal feed. In this paper, an exact and robust analysis method is described for the quantification of phorbol esters (PE) in Jatropha curcas L. seed oil. This method procedure includes the extraction of PE in methanol, chromatographic separation on a reverse phase C18 HPLC column and the quantification by standard addition method. For the standard addition method a highly pure PE standard is used, which is extracted and purified by semi preparative HPLC right before the measurements. The used detector for identification and quantification is UV set at 280 nm and ESI‐ToF‐MS with a ±10 ppm mass difference of the deprotonated and formate adduct pseudo molecular ion of PE.

     

Fully Automated Three-Dimensional Column-Switching SPE–FIA–HPLC System for the Characterization of Lipids by a Single Injection: Part I. Instrumental Design and Chemometric Approach to Assess the Effect of Experimental Settings on the Response of ELSD

This article presents the first application of fully automated three‐dimensional (3D) column‐switching SPE–FIA–HPLC system for the characterization of lipids by a single injection. The whole system was designed and set up by modifying Agilent 1200 Series HPLC system in our laboratory. By using this system, a complete separation profile of the oil samples was achieved in a very short time period by using single injections. This approach was applied on vegetable oils which contains a large number of relatively high‐class lipid components, such as TG, FFA, sterols, tocopherols, DG, ester and MG. In this part of the study, we focused on the optimization of evaporative light scattering detector (ELSD) by using an experimental design and RSM. Three experimental parameters were chosen as an independent variables which are the flow rate of mobile phase, nebulization temperature and evaporation temperature. A multivariate five level experimental design was used to establish a quadratic model as a functional relationship between the response values and independent variables. The optimal values of parameters were found to be a flow rate of 1.25 mL min^?1, nebulization temperature of 80 °C, and evaporation temperature of 40 °C. Regression analysis with an R² values indicated as a satisfactory correlation between the experimental and predicted values. ANOVA test results were also illustrate that the models can be successfully used to predict the optimum parameters of ELSD. Thus, the proposed system is suitable for a large number of applications including research and development of new quality control and characterization methods for vegetable oils.     

Determination of optical purity and absolute configuration of natural 12‐hydroxystearic acid by the 1H NMR anisotropy method

The enantiopure of (R)‐(?) MαNPA was allowed to react with racemic 18‐(tert‐butyldimethylsilyloxy)‐5‐octadecayne‐7‐ol which was derived from dodecane‐1,12‐diol, yielding diastereomeric esters mixture. These racemic esters were easily separated by HPLC on silica‐gel. The absolute configurations of the first‐eluted diastereomeric esters from the separated esters were determined using ¹H NMR anisotropy method. Analysis on ¹H NMR spectra and HPLC elution time of the synthesized esters and those of MαNP ester derived from natural methyl‐12‐hydroxystealate showed that the absolute configuration of natural 12‐hydroxystearic acid was R form, and the enantiomeric excess was over 99%.     

Effect of nano‐silica on gas permeation properties of polyether‐based polyurethane membrane in the presence of esterified canola oil diol as a nucleation agent for hard segments

In this research esterified canola oil diol (COD) was used to synthesize a green thermoplastic polyurethane. The mixture of synthesized COD as a polyester and polytetramethylene‐glycol as a polyether with different molar ratios were used to synthesize a thermoplastic polyurethane. Membranes were prepared by solution casting technique and nano‐silica particles were used to improve their gas separation performance. The effects of COD segments on phase separation and thermal properties of blocky segments of polyurethanes were evaluated using Fourier transform infrared spectroscopy and thermal gravimetric analysis. Results showed that phase separation behavior of the synthesized polyurethane was significantly increased with COD content. The COD segments showed high tendency to interact with hard segments of polyurethane in a way that new domains with higher thermal stability is created. Permeability of pure CO₂, CH₄, N₂, and He gases were taken using constant pressure method at different pressures. Nano‐silica particles showed high inclination to interact with COD segments and significantly influenced the phase separation as well as gas permeation properties of polyurethane. Interactions of nano‐silica particles with the soft segments of polyurethane increased the glassy behavior of polymer and improved the CO₂/CH₄, CO₂/N₂, and CO₂/He ideal selectivities (permselectivities). © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 45979.     

Separation and Quantification of Vegetable Oil Based Polyols by High Performance Liquid Chromatography with Evaporative Light Scattering Detection

Polyols with terminal primary alcohol functionalities were obtained from canola oil via an ozonolysis and hydrogenation process. A high performance liquid chromatography (HPLC) method with evaporative light scattering detection (ELSD) was developed for separating and quantifying the polyol products. Linear calibration curves were obtained for the mono-ol, diol and triol components with correlations (r ²) above 0.98. According to the standard curve, the content of mono-ol, diol and triol can be obtained from their HPLC-ELSD chromatograms.     

Rapid Detection and Quantification of Triacylglycerol by HPLC–ELSD in Chlamydomonas reinhardtii and Chlorella Strains

Triacylglycerol (TAG) analysis and quantification are commonly performed by first obtaining a purified TAG fraction from a total neutral lipid extract using thin-layer chromatography (TLC), and then analyzing the fatty acid composition of the purified TAG fraction by gas chromatography (GC). This process is time-consuming, labor intensive and is not suitable for analysis of small sample sizes or large numbers. A rapid and efficient method for monitoring oil accumulation in algae using high performance liquid chromatography for separation of all lipid classes combined with detection by evaporative light scattering (HPLC–ELSD) was developed and compared to the conventional TLC/GC method. TAG accumulation in two Chlamydomonas reinhardtii (21 gr and CC503) and three Chlorella strains (UTEX 1230, CS01 and UTEX 2229) grown under conditions of nitrogen depletion was measured. The TAG levels were found to be 3–6 % DW (Chlamydomonas strains) and 7–12 % DW (Chlorella strains) respectively by both HPLC–ELSD and TLC/GC methods. HPLC–ELSD resolved the major lipid classes such as carotenoids, TAG, diacylglycerol (DAG), free fatty acids, phospholipids, and galactolipids in a 15-min run. Quantitation of TAG content was based on comparison to calibration curves of trihexadecanoin (16:0 TAG) and trioctadecadienoin (18:2 TAG) and showed linearity from 0.2 to 10 μg. Algal TAG levels >0.5 μg/g DW were detectable by this method. Furthermore TAG content in Chlorella kessleri UTEX 2229 could be detected. TAG as well as DAG and TAG content were estimated at 1.6 % DW by HPLC–ELSD, while it was undetectable by TLC/GC method.     

Rapid determination of cholesterol in emulsified confectioneries by ultra‐performance liquid chromatography

We developed a sample preparation method by using no‐heating saponification after dispersive SPE and ultra‐performance liquid chromatography for rapid determination of cholesterol in emulsified foods, such as cream pie, cream sandwich‐type biscuit, plate chocolate, and chocobar. Optimum cholesterol extraction was obtained by using 10 mL isopropyl alcohol and 8.0 g (NH₄)₂SO₄ per 1–2 g sample; saponification after extraction was performed using 25 mg KOH, 0.8 g NaCl, and 100 mg of a silica based NH₂ SPE material. The determined cholesterol concentration was within certified reference material limits, and results of the recovery test were in the range of 97.4 to 104.8% with a relative standard deviation between 2.14 and 3.80%. Since our method had less sample preparation and chromatographic separation times, we expect our method to be used for effective quality control processes for monitoring cholesterol concentrations in confectioneries. Practical applications: A rapid analytical method for determining the cholesterol concentration in emulsified confectioneries was developed by using UPLC. The assessed cholesterol levels after comparing them with the CRMs were within the permissible range; results of the recovery tests performed using spiked materials ranged from 97.4 to 104.8% with RSD between 2.14 and 3.80%. Thus, our method could be used for accurate and precise determination of cholesterol concentration at low analysis and pretreatment time.     

Preparation of stearidonic acid‐enriched triacylglycerols from Echium plantagineum seed oil

HPLC analysis of Echium plantagineum seed oil shows a complex triacylglycerol (TAG) profile. TAG species were separated on an analytical scale by HPLC and their fatty acid (FA) composition is reported. GLC analyses showed that some TAG fractions reached a stearidonic acid (SDA, 18:4n‐3) percentage significantly higher than that in the original oil. TAG separation on a bigger scale was also essayed, by means of a gravimetric normal‐phase chromatographic column, using silver ion‐silica gel as stationary phase. Gradient elution with solvents of increasing polarity was applied, allowing the separation of valuable TAG species containing γ‐linolenic acid (GLA, 18:3n‐6), α‐linolenic acid (ALA, 18:3n‐3) and SDA as the main constituents (more than 85% of the total FA). An enzymatic hydrolysis reaction showed the distribution of FA in the isolated species of TAG. SDA was the major FA in the sn‐2 position (more than 50% of total FA), followed by ALA (19%) and GLA (18.5%).     

Cyclic fatty acid monomer: Isolation and purification with solid phase extraction

The application of a combined solid phase extraction (SPE)/clean-up procedure to the isolation of a purified fraction containing all the monomeric cyclic fatty acid methyl esters is described. Extraction of the nonpolar lipid components from nonurea-adducting (NUA) filtrates is performed on a reverse phase octadecyl bonded silica minicolumn. Stepwise elution of the SPE-retained materials through silica gel using several solvents allowed the separation of a pure fraction containing the cyclic monomers that can be used for a more reliable quantitative estimation of these compounds in edible fats and oils.     

In situ FT‐IR‐ATR studies on the structure development of polyurethane‐urea systems

This article reports the application of Fourier Transform Infrared‐Attenuated Total Reflectance (FTIR‐ATR) technique for investigation of in situ polymerization of polyurethane‐urea elastomers. Formulations comprising of diisocyanates, a polyether diol and a diamine based chain extender have been used in this study. The diisocyanates used were 4,4′‐diphenylmethane diisocyanate and toluene diisocyanates. The polyether diol and diamine used were propyleneglycol block‐PEO‐b‐PPO and 4‐(4‐(2‐(4‐(4‐amino‐2‐(trifluoromethyl) phenoxy) phenyl) propan‐2‐yl) phenoxy)‐3‐(trifluoromethyl) benzeneamine, respectively. These reactants were mixed and placed on the ATR cell, and then the infrared spectra were recorded at an interval of 1.75 s while continuously heating. The polyurethane‐urea formation was monitored by the decay in the intensity of isocyanate band at 2258 to 2261 cm^?1. As the polymerization progressed, new peaks appeared at wavenumbers of 1711 cm^?1, 1697 cm^?1, and 1655 cm^?1. These peaks correspond to the urethane carbonyl, hydrogen bonded urethane, and urea carbonyl groups, respectively. It was found that with the progress of the reaction, the shift in the peaks at 1655 and 1697 cm^?1 occurs gradually. This shift in peaks is attributed to the hydrogen bonding. The hydrogen bonding and hence the shift in the peak is a cumulative effect of three phenomena namely: (1) degree of polymerization, (2) macro and microphase separation, and (3) temperature effect. A rationale is discussed to deconvolute these three effects. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Improvement of accuracy in quantification of 3‐monochloropropane‐1,2‐diol esters by Deutsche Gesellschaft für Fettwissenschaft standard methods C‐III 18

By Deutsche Gesellschaft für Fettwissenschaft (DGF) standard methods C‐III 18 for the determination of 3‐monochloropropane‐1,2‐diol (3‐MCPD), the minimum limit of detection was lower in the case of actual oil samples compared to the calibration samples. The problem was found to be lied in the low recovery of 3‐MCPD derivatives from the aqueous phase to the organic phase at the extraction step of the standard procedure. The substitution of the conventional solvent, n‐hexane, with n‐butanol, chloroform, and ethyl acetate increased the recovery to the relative extent of 5.6, 4.7, and 3.9, respectively. The modification contributed to improve the accuracy of the method, especially at lower concentration (<1 ppm) of 3‐MCPD. Practical applications: This paper provides the modification of DGF standard methods C‐III 18 in order to improve the accuracy to quantify 3‐MCPD at lower concentration. It might be important for estimation and control of our daily intake of 3‐MCPD, and for the product control in the fat and oil processing.     

Synthesis and Reactions of Enantiopure Substituted Benzene cis‐Hexahydro‐1,2‐diols

Enantiopure cis‐dihydro‐1,2‐diol metabolites, obtained from toluene dioxygenase‐catalysed cis‐dihydroxylation of six monosubstituted benzene substrates, have been converted to their corresponding cis‐hexahydro‐1,2‐diol derivatives by catalytic hydrogenation via their cis‐tetrahydro‐1,2‐diol intermediates. Optimal reaction conditions for total catalytic hydrogenation of the cis‐dihydro‐1,2‐diols have been established using six heterogeneous catalysts. The relative and absolute configurations of the resulting benzene cis‐hexahydro‐1,2‐diol products have been unequivocally established by X‐ray crystallography and NMR spectroscopy. Methods have been developed to obtain enantiopure cis‐hexahydro‐1,2‐diol diastereoisomers, to desymmetrise a meso‐cis‐hexahydro‐1,2‐diol and to synthesise 2‐substituted cyclohexanols. The potential of these enantiopure cyclohexanols as chiral reagents was briefly evaluated through their application in the synthesis of two enantiomerically enriched phosphine oxides from the corresponding racemic phosphine precursors.     

Oxidation products of free polyunsaturated fatty acids in wheat varieties

Oxygenated fatty acids (oxylipins) are secondary metabolites of polyunsaturated fatty acids (PUFA). Here, we present a novel high‐performance liquid chromatograpic separation on a reversed‐phase column (RP‐HPLC) coupled with electrospray ionization‐tandem mass spectrometry (ESI‐MS/MS) for the determination of various (per)oxidation products of linoleic (cis,cis‐9,12‐octadecadienoic) acid in eight different varieties (four spring and four winter varieties) of wheat (Triticum aestivum). The procedure includes extraction of oxylipins, chromatograpic separation using a linear gradient of aqueous formic acid and acetonitrile, with subsequent identification of compounds by MS/MS. Among the identified oxylipins, leukotoxin (LTX)‐diol and its isomer (iso‐LTX‐diol) are known as potentially toxic substances. The obtained data was used further for comparison of different wheat varieties by principal component analysis (PCA). From the results of PCA, differences can be observed in the patterns of wheat varieties.     

Analysis of insect triacylglycerols using liquid chromatography‐atmospheric pressure chemical ionization‐mass spectrometry

An HPLC non‐aqueous reversed‐phase separation system was adapted for analyzing insect triacylglycerols (TAG). The method uses two conventional Nova‐Pak C18 columns connected in series, for a total length of 45 cm. The mobile phase gradient is mixed from acetonitrile and 2‐propanol, and the flow rate is programmed from 1.0 to 0.7 mL/min. TAG are detected by atmospheric pressure chemical ionization‐mass spectrometry. The method ensures efficient separation of isomers and analysis of high‐molecular‐weight TAG with equivalent chain lengths up to 72. The method performance is demonstrated on analysis of TAG isolated from the fat body of the bumblebee Bombus lucorum.