Postcolumn fluorescence as an alternative to evaporative light scattering detection for ceramide analysis with gradient elution in non-aqueous reversed-phase liquid chromatography

Ceramide analysis was developed with gradient elution in non-aqueous reversed-phase liquid chromatography with evaporative light scattering detection (ELSD) or postcolumn fluorescence detection. Fluorescence detection (excitation, 360 nm; emission, 425 nm) after postcolumn formation of mixed assemblies between eluted ceramides and 1,6-diphenyl-1,3,5-hexatriene was developed. In comparison with ELSD, fluorescence detection allows a better detection of the minor species ceramide from ceramide type III (commercial mixture of non-hydroxy fatty acid-sphingosine) and appears to be more sensitive for quantitation of ceramides at low concentrations. The fluorescence response is linear over a wide range of injected amount of ceramide III (expressed as stearoyl-phytosphingosine): 10 ng to 1000 ng. The response of ELSD is non linear but can be linearized in double logarithmic coordinates for calculations over a narrow range, e.g. between 10 to 350 ng ceramide III injected. The lower quantitation limits of these two detectors are similar: 5 ng ceramide III was injected.     

Microanalytical systems for separations of stratum corneum ceramides

The small amount of lipids from human skin obtained with noninvasive sampling method led us to investigate microanalytical separation techniques. The lipid class analysis was performed with a micro polyvinyl alcohol-silica (PVA-Sil) column. The gradient elution was from heptane to acetone/butanol 90:10 v/v in 4%/min at 78 microL/min. In addition an evaporative light scattering detector (ELSD) was modified for micro-LC. All solvents contained 0.1% of triethylamine and formic acid in stoichiometric amount, which increased the ELSD response. In these conditions, the cholesterol eluted before free fatty acid, and squalene and triglycerides close to the dead volume. The various ceramide classes eluted following the order of the increased number of hydroxyl groups. The LOD for ceramides was 2.2 ng. The advantages of this method are the use of a normal stationary phase more reliable due to its chemical stability, its surface homogeneity and its development in microchromatography without chlorinated solvents which offers small LOD and the whole profile of lipids present in stratum corneum (SC). A method using a narrow-bore PVA-Sil column was used to collect ceramide fraction. Then the molecular species were analysed with a porous graphitic carbon column in capillary LC using a gradient from CH3OH/CHCl3 70:30 v/v to CHCl3 at 2%/min with a flow rate at 5 microL/min. The LOD obtained for ceramide was 1 ng. Both methods were assessed with SC samples obtained by rinsing a 5.7 cm2 area of the forearm with 25 mL of ethanol.     

Evaluation of a pulsed EIMAC source for atomic-fluorescence spectrometry

An EIMAC xenon arc continuum source operated in the pulsed mode has been evaluated for potential use as a source of excitation in atomic-fluorescence spectrometry. The results were compared with those obtained with a similar lamp operated in the continuous-wave mode; little or no improvement was evident from this comparison. The lamp was found to be unstable in the pulsed mode, requiring the use of a rather large residual d.c. current in conjunction with a relatively low amplitude power-pulse in order to sustain the arc. Moreover, the pulsed source yielded a large background signal because of scattering of source-radiation in the flame; the background spectrum (with source "on") contained numerous large peaks due to fluorescence of molecular species in the flame and to irregularities in the spectral distribution of the pulsed lamp output transmitted to the signal detector by scatter.     

Construction of a light-emitting diode fluorescence detector for high-performance liquid chromatography and its application to fluorometric determination of L-3-hydroxybutyrate

This study employed a new light source, a light-emitting diode (LED), for fluorescence detection of high-performance liquid chromatography to measure the concentration of trace constituents in biological fluids. Using l-3-hydroxybutyrate ( l-3HB) as a tested trace compound, the function of the new system was compared with that of the current commercially available model. A detailed schematic diagram of the path of the detection rays in the LED detector is given. A voltage-stabilizer for the drive circuit was designed with an input of 10 V and an output of 8 V, and another voltage regulator was used to maintain a constant 8 V. Then the regulator was used to set the output voltage for the LED at 2.8 V by two external resistors. Replacing the xenon lamp with LED, this system provided higher photon density and a narrow spectrum at a wavelength of 491 nm. At room temperature (22.1°C), the average temperature of six places in the chamber of LED detector was 22.1°C compared with 51.1°C in the xenon detector. The spectra of the excitation light sources were measured. Compared with the xenon lamp, approximately 1.32 times higher excitation intensity was obtained by the LED source. The accuracy of detection of l-3HB in 50 μL of rat serum was 99.85-100.85%, and the intra-day and inter-day precision values were within 8.99 and 13.90%, respectively. The limit of detection of l-3HB was approximately 0.73 μM (signal-to-noise ratio 3). The sensitivity of the proposed LED detector was comparable to that of traditional fluorescence detectors using xenon arc lamps; however, the cost and operating temperature of LED lamps were far lower. This assay system could be further used to detect trace constituents in various samples.     

High-temperature micro liquid chromatography for lipid molecular species analysis with evaporative light scattering detection

The need for a rapid and sensitive chromatographic technique for analyzing lipid molecular species, has led to the development of an high-temperature micro liquid chromatographic system (HTLC) coupled to an evaporative light scattering detector. The increased diffusion coefficients and reduced viscosity at higher temperatures allowed lipids to be analyzed rapidly with solvents differing from those classically used in lipids chemistry. Hypercarb, a reverse phase material, was used for its different properties including heat resistance in high temperature micro HPLC. We have investigated the temperature effect on kinetic performances in HTLC, established pure solvents eluent strength at high temperature and studied different classes of lipids with seven pure solvents. We found that it was possible to use alcohols solvents in the mobile phase to elute lipids without the use of chlorinated solvents. A quick and simple method was developed to analyze a complex lipid simple, ceramide type III and type IV.     

Anomalies in evaporative light scattering detection

A two-dimensional (2-D) “heart-cutting” HPLC system was used to fractionate oligostyrenes into the respective diastereoisomers. For samples of known composition, the response of an ultraviolet (UV) absorbance detector followed the anticipated pattern. The response of an evaporative light-scattering (ELSD) detector on the other hand indicated quite different concentrations for the two diastereoisomers, relative to what was anticipated and what was indicated by the UV detector. Whereas approximately the same concentration was indicated by UV, ELSD in some cases indicated no detection of the later eluting isomer. The magnitude of the errors depended on both the molecular weight and the tacticity of the diastereomers. These anomalies appear to be an artifact of power transform functions imbedded within the firmware processor of the ELSD, invisible to the user.     

Comparative study of photodegradation of wood by a UV laser and a xenon light source

A detailed study of photodegradation of wood surfaces by xenon light source and a UV laser has been carried out. Silver birch, rubberwood, Scots pine and chir pine wood veneers were irradiated with a xenon light source or a 244 nm argon ion laser. The changes in chemical structure of wood surfaces were monitored by UV resonance Raman (UVRR), photoacoustic Fourier transform infrared (FTIR-PAS) and UV-vis reflectance spectroscopies. The depth profile of xenon lamp irradiated wood surfaces was carried out by measuring FTIR-PAS spectra at different moving mirror velocities. The UVRR and FTIR-PAS spectra of irradiated wood surfaces showed degradation of aromatic structure in lignin combined with strong formation of carbonyl structures. The FTIR-PAS spectra measured from xenon irradiated wood surfaces indicate that hardwood lignin degrades at a faster rate than softwood lignin. The UVRR spectra of xenon irradiated wood show a significant decrease in the intensities of aromatic structures at 1602 cm⁻¹. This is accompanied by a significant band broadening and notable shift towards longer wavenumbers, which has been attributed to the formation of o- and p-quinone structures as degradation products. The formation of quinone structures was also supported by the generation of a broad absorption band between 350 and 600 nm in UV-vis reflectance spectra of irradiated wood surfaces. There was a significant broadening in the region of 1500-1000 cm⁻¹ in UVRR spectra due to the formation of unsaturated structures as a result of lignin degradation. The UVRR spectra of laser irradiated wood showed similar behaviour i.e., overall broadening and a rapid reduction in the intensity of lignin aromatic structure. The rate of degradation by laser was very high. However, the extent of band broadening was higher in xenon irradiated wood indicating the generation of several different types of structures as compared to laser irradiation, which produces only particular type of structures. UVRR spectra of laser irradiated Whatman paper showed significant photodegradation of cellulose by UV laser. The UV degradation rate of lignin was much higher than cellulose.     

Performance evaluation of evaporative light scattering detection and charged aerosol detection in reversed phase liquid chromatography

In pharmaceutical industry ultraviolet (UV) detection is often used as the preferred detection technique in HPLC analysis since most pharmaceutical compounds possess a UV-absorbing chromophore. However, in case the active pharmaceutical ingredient (API) does not have a UV-absorbing chromophore, or if some of the impurities present lack a chromophore, they will not be detected in routine HPLC analysis employing only a UV detector and alternative detection schemes have to be used. Refractive index detection or mass spectroscopy (MS) can be used but these detectors have their intrinsic weaknesses, such as lack of sensitivity or high cost. With the appearance of semi-universal techniques such as evaporative light scattering detection (ELSD), and more recent, charged aerosol detection (CAD), detection of non-UV-absorbing compounds became feasible without having to resort to such complex or costly detection methods. This paper evaluates the different performance characteristics such as sensitivity, linearity, accuracy and precision of both the ELSD and CAD detector coupled to HPLC. One disadvantage of this type of detector is the non-linear response behaviour which makes direct linear regression for making calibration curves inaccurate.     

Inductively coupled plasma-ionic absorption spectrometry using a xenon arc lamp and an echelle monochromator

Ionic absorption spectrometry using the inductively coupled plasma (ICP) as an ionization source and a 150 W stable xenon arc lamp as a light source has been investigated. A high-resolution echelle spectrometer was used for the absorption measurement. Ionic absorption lines in the wavelength region above 320 nm showed high sensitivity and detection limits of Y, Ti, Zr, Sc, Ce and La were of the same order of magnitude as those attained by ICP atomic emission spectrometry. Linear dynamic ranges of three orders of magnitude were observed for most elements tested. Effects of the measurement conditions such as rf power, outer gas flow rate, inner gas flow rate and observation height on the transmittance of the beam through the plasma and the absorbed radiation power of the analytes are also given.     

Determination of Biodiesel and Triacylglycerols in Diesel Fuel by LC

A high performance liquid chromatographic method was developed for quantifying blends of biodiesel (simple alkyl esters of fatty acids) in petrodiesel. The method uses a silica column with an isocratic mobile phase consisting of hexane and methyl t-butyl ether. Separated components were quantitated using either an evaporative light scattering detector (ELSD) or UV detector. Precision of injection and linearity of response of the ELSD and UV detectors over a range of biodiesel-petrodiesel blends [1–30 v/v %] were established by use of standards. The method also can be used for quantitating similar levels of oils or fats (triacylglycerols) in petrodiesel.     

Effects of different light irradiations on structure and optical properties of methoxy-substituted tetraphenylporphyrins

UV lamp, filtered halogen lamp (at 425 nm) and Green laser (532 nm) experiments on a series of meso-substituted tetra phenyl porphyrin, TPP, bearing methoxy peripheral groups together with a metal derivate of 3,4 dimethoxy TPP were lead to different protonation and aggregation structures. Properties of irradiated porphyrins were investigated using their absorption and emission spectra in dichloromethane solution. The results show that the optical properties of the TPP derivates depend on light irradiation source, which shows the tuning of the absorption and emission spectra of the TPP derivates. From the dynamic light scattering measurements, the size distribution of samples was estimated about 5–15 nm in solvent after irradiation. Atomic force microscopy images of deposited porphyrins on the glass surface were shown average particle size between 10 and 30 nm. Particularly, self-assembly of the porphyrin derivates was also observed when green laser was used. We suggest that the irradiation source plays an important role in the controlling of size and morphology of products, and we propose a self-organization model to explain the formation of the porphyrin nanostructures.     

Comparison between evaporative light scattering detection and charged aerosol detection for the analysis of saikosaponins

Saikosaponins are triterpene saponins derived from the roots of Bupleurum falcatum L. (Umbelliferae), which has been traditionally used to treat fever, inflammation, liver diseases, and nephritis. It is difficult to analyze saikosaponins using HPLC-UV due to the lack of chromophores. Therefore, evaporative light scattering detection (ELSD) is used as a valuable alternative to UV detection. More recently, a charged aerosol detection (CAD) method has been developed to improve the sensitivity and reproducibility of ELSD. In this study, we compared CAD and ELSD methods in the simultaneous analysis of 10 saikosaponins, including saikosaponins-A, -B₁, -B₂, -B₃, -B₄, -C, -D, -G, -H and -I. A mixture of the 10 saikosaponins was injected into the Ascentis^® Express C18 column (100 mm × 4.6 mm, 2.7 μm) with gradient elution and detection with CAD and ELSD by splitting. We examined various factors that could affect the sensitivity of the detectors including various concentrations of additives, pH and flow rate of the mobile phase, purity of nitrogen gas and the CAD range. The sensitivity was determined based on the signal-to-noise ratio. The best sensitivity for CAD was achieved with 0.1 mM ammonium acetate at pH 4.0 in the mobile phase with a flow rate of 1.0 mL/min, and the CAD range at 100 pA, whereas that for ELSD was achieved with 0.01% acetic acid in the mobile phase with a flow rate at 0.8 mL/min. The purity of the nitrogen gas had only minor effects on the sensitivities of both detectors. Finally, the sensitivity for CAD was two to six times better than that of ELSD. Taken together, these results suggest that CAD provides a more sensitive analysis of the 10 saikosaponins than does ELSD.     

Evaporative light scattering detection based HPLC method for the determination of polysorbate 80 in therapeutic protein formulations

An evaporative light scattering detection (ELSD) based high-performance liquid chromatography (HPLC) method is developed for the determination of polysorbate 80 (tween 80) in therapeutic protein formulations. The method is simple and overcomes the difficulties associated with specificity and sensitivity. The method is suitable for the quantitation of polysorbate 80 in the usual formulation range (0.01-0.1%) as well as in trace amounts ≥13 μg/mL. The analysis is based on the removal of protein first by solid-phase extraction using Oasis HLB cartridges followed by HPLC analysis using Inertsil ODS-3 C 18 column (4.6×150 mm, 5 μm) using reversed-phase conditions. The detector response changes exponentially with an increase in polysorbate concentration. A very good linear fit of log ELSD response against log polysorbate 80 concentration is observed. The specificity, sensitivity, precision, and accuracy of the method are suitable for the quantitation of polysorbate 80 in protein formulations.     

Decomposition of organic compounds by irradiation with light

Summary It has been shown that organic halogen compounds completely decompose under the light of a xenon lamp. Decomposition products include hydrogen hah'de, free halogen, free carbon, carbon monoxide, gaseous hydrogen. The rate and completeness of the photo-decay depends on the nature of both the compound and the halogen. The minimum time of decay was observed for iodo-derivatives, the maximum for fluoro-derivatives. The rate of photo-decay was found to be considerably greater for coloured substances.

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Zusammenfassung Organische Halogenverbindungen zersetzen sich im Licht einer Xenonlampe. Unter den Zersetzungsprodukten finden sich Halogenwasserstoff, freies Halogen, freier Kohlenstoff, Kohlenmonoxid und gasförmiger Wasserstoff. Das Ausmaß und die Vollständigkeit der Photozersetzung hängt von der Art der Verbindung und der Art des Halogens ab. Die kürzeste Zersetzungszeit wurde bei Jodderivaten, die längste bei Fluorderivaten beobachtet. Die Geschwindigkeit der Zersetzung ist bei gefärbten Substanzen erheblich größer.

     

Determination of gentamicin sulfate and related compounds by high-performance liquid chromatography with evaporative light scattering detection

A rapid and simple method for the separation and quantitation of gentamicin sulfate by HPLC coupled with evaporative light scattering detection (ELSD) has been developed. Detection of the different components of gentamicin is problematic because of the lack of UV absorbing chromophore. The use of the universal ELSD avoids the need for sample derivatization or use of specific detector such as pulsed amperometry. Separation was performed on a highpurity C18 125 mm x 4 mm i.d., 3 microm, reversed phase column with 48.5 mM trifluoroacetic acid-methanol (97:3, v/v), as mobile phase at a flow rate of 0.7 ml/min. The influence of the gas nature, gas pressure and temperature of the drift tube of the detector on the detection response was investigated. Optimization was performed with the help of a specific experimental design software. This method allows the determination of the composition in components C1, C1a, C2, C2a and C2b of gentamicin sulfate samples. Mass spectrometry was employed to confirm the ELSD chromatographic profile. The method was validated using methodology described by the International Conference of Harmonization in the field of Medicinal Substances. Commercial samples of different sources were analyzed and results were in good agreement with specifications of both European and United States Pharmacopoeia.     

Recent developments with light-exposure apparatus and measurement of radiation of their light sources

New techniques and devices which were recently developed in order to avoid various drawbacks of current light-exposure apparatus are introduced. The spectral distribution of the open flame carbon arc approximates more closely to that of sunlight by modifications of the filter system.The life of carbon arcs was lengthened more than two fold. The long-life xenon arc lamp minimized the reduction in output of radiation.The slanted holder improved the locational variance of radiation falling on a specimen.The instrumental methods for measuring light dosage are explained.     

Evaluation of the coupling between ultra performance liquid chromatography and evaporative light scattering detector for selected phytochemical applications

Fast analysis in LC can be performed with sub-2 microm particles at very high pressures (up to 1000 bar) known as ultra performance LC (UPLC). With this configuration, it is possible to obtain fast and/or highly efficient separations compared to conventional LC. For the analysis of compounds without chromophores, the evaporative light scattering detector (ELSD) is an attractive alternative because of its quasi-universality, versatility, low-cost and good sensitivity. The UPLC-ELSD was investigated in terms of sensitivity and apparent efficiency, with a conventional ELSD instrument, for two types of commercially available nebulisers, using different mobile phase flow rates and column ids. Results were finally compared with the UPLC-UV configuration. Three applications with phytochemical compounds were selected to highlight the potential of this approach (i.e. the isocratic separations of artemisinin and its derivatives, of calystegines and the gradient separation of several tropane alkaloids). Depending on the used column length, baseline separations were obtained in 3-10 min, with an average apparent efficiency ranging from 7000 to 30,000 plates.     

Mechanism of response enhancement in evaporative light scattering detection with the addition of triethylamine and formic acid

Triethylamine with an equimolar amount of formic acid added to the mobile phase provides an enhancement of the evaporative light scattering detector (ELSD) response. After characterization of the influence of various parameters on the ELSD response, a sequential strategy was defined to elucidate this response enhancement. The response enhancement was more marked at low mobile phase flow rate, and was highly dependent on solutes and solvents. The influence of drift tube temperature on response enhancement with various solutes demonstrated that triethylamine and formic acid mainly act as mass amplifiers by the inclusion of triethylamine-formic acid clusters inside the droplets.     

聚丙烯/纳米级金红石型二氧化钛/聚烯烃弹性体复合材料的抗老化性能研究

采用TEM和UV-Vis等测试手段表征了金红石型纳米级TiO2和体相TiO2的性能特征;通过熔融共混法分别制备了PP/纳米级TiO2/POE和PP/体相TiO2/POE复合材料,采用GB/T16422·2-1999所述的塑料实验室光源暴露实验方法,用氙灯气候试验机对纯PP和复合材料进行28天人工加速老化.结果表明,二氧化钛粒子在PP/POE基体中分散性良好,而纳米粒子对PP/POE基体具有增韧作用;改性后的两类复合材料均具有优异的抗老化性能,而PP/(1·0wt%)纳米级TiO2/POE复合材料的抗老化性能更加优异,其加速老化28天后的无缺口冲击强度达到80·45kJ·m-2,比纯PP提高4倍多,而同期加速老化28天后的PP/(1·0wt%)体相TiO2/POE复合材料的无缺口冲击强度只有47·88kJ·m-2;对纯PP老化过程中的羰基指数和冲击性能的变化情况进行了分析,发现二者近似成线性关系,其相关系数r在0·9以上.     

Analysis of phenolic glycosides and saponins in Primula elatior and Primula veris (primula root) by liquid chromatography, evaporative light scattering detection and mass spectrometry

This paper describes the first liquid chromatographic method suitable for the simultaneous determination of bioactive compounds, saponins and phenolic glycosides, present in Primula elatior and Primula veris, including the NMR data of primulaverin and primeverin. Optimum separations were obtained with a Synergi 4mum Fusion RP 80A column, using 0.025% TFA in water and 5% acetonitrile in methanol as mobile phase. Saponins were detected by evaporative light scattering detection (ELSD), whereas the phenolic glycosides were monitored by UV at 210 nm. The method was validated for repeatability (sigma(rel) or=97.1%) and sensitivity (LOD 相似文献