Thin-layer chromatography combined with MALDI-TOF-MS and 31P-NMR to study possible selective bindings of phospholipids to silica gel

High-performance thin-layer chromatography (HPTLC) is a highly established separation method in the field of lipid and (particularly) phospholipid (PL) research. HPTLC is not only used to identify certain lipids in a mixture but also to isolate lipids (preparative TLC). To do this, the lipids are separated and subsequently re-eluted from the silica gel. Unfortunately, it is not yet known whether all PLs are eluted to the same extent or whether some lipids bind selectively to the silica gel. It is also not known whether differences in the fatty acyl compositions affect the affinities to the stationary phase. We have tried to clarify these questions by using a readily available extract from hen egg yolk as a selected example of a lipid mixture. After separation, the complete lanes or selected spots were eluted from the silica gel and investigated by a combination of MALDI-TOF MS and ³¹P NMR spectroscopy. The data obtained were compared with the composition of the total extract (without HPTLC). Although there were significant, solvent-dependent losses in the amount of each lipid, the relative composition of the mixture remained constant; there were also only very slight changes in the fatty acyl compositions of the individual PL classes. Therefore, lipid isolation by TLC may be used without any risk of major sample alterations.

High-resolution monolithic columns—a new tool for effective and quick separation

This work describes a comparison of three types of commercial high-performance liquid chromatography silica monolithic columns with different inner diameters and generations of monolithic sorbent: a “classic” monolithic column, the first generation (Onyx? monolithic C₁₈, 100 mm?×?4.6 mm, Phenomenex); a “narrow” monolithic column for fast separation at lower flow rates (Chromolith® Performance RP-18e, 100 mm?×?3 mm, Merck); and a recently introduced “high-resolution” monolithic column, the next generation (Chromolith® HighResolution RP-18e, 100 mm?×?4.6 mm, Merck). Separation efficiency (number of theoretical plates, height equivalent to a theoretical plate and van Deemter curves), working pressure, the symmetry factor and resolution were critical aspects of the comparison in the case of the separation of ascorbic acid, paracetamol and caffeine. The separations were performed under isocratic conditions with a mobile phase consisting of 10:90 (v/v) acetonitrile–phosphoric acid (pH 2.80). Detailed comparison of the newest-generation monolithic column (Chromolith® HighResolution) with the previously introduced monolithic sorbents was performed and proved the advantages of the Chromolith® HighResolution column.

Oriented immobilisation of histidine-tagged protein and its application in exploring interactions between ligands and proteins

A method based on reaction with a diazonium salt was developed to immobilise oriented His-tagged protein onto silica gel. The binding efficiency of the phenylamine-group-coated gel was determined to be 65 %, providing a binding capacity of His-tagged protein up to the gram level. Using His-tagged β₂-adrenoceptor (β₂-AR) as a probe, we developed a new mathematical model to elucidate the interactions between the receptor and five ligands (methoxyphenamine, terbutaline, salbutamol, tulobuterol and fenoterol). These drugs proved to only have one type of binding site on the immobilised β₂-AR, yielding higher association constants and numbers of binding sites than random attachment assays. The association constants determined by the new model positively correlated to the values from a radioligand binding method, with a regression equation of y?=?1.75x???7.18 and a correlation coefficient of 0.9807. The oriented method resulted in a high binding capacity and quantitative immobilisation of the His-tagged protein. The proposed model can be used to determine the interactions between the ligands and the immobilised protein with the advantages of drug and time saving.

Solid phase ligand-less extraction of cadmium(II) using a silica gel modified with an amino-functionalized ionic liquid

A method was developed for the determination of cadmium(II) by ligand-less solid phase extraction that is based on the direct retention of Cd(II) in a mini-column filled with a silica gel modified with an amino-functionalized ionic liquid. The effects of pH, sample volume and its flow rate, eluent concentration and its volume, the flow rate of eluent, and of potential interferences on extraction and desorption were optimized. Following its determination by electrothermal atomic absorption spectrometry, the detection limit for Cd(II) is 8.9 ng L^?1, and the relative standard deviation is 2.3 % (at 1.0 ng mL^?1; for n?=?5). The method was applied to the analysis of Cd(II) in a certified reference material (laver; GBW10023), and the recoveries ranged from 97.0 to104.0 %

Reagentless d-sorbitol biosensor based on d-sorbitol dehydrogenase immobilized in a sol–gel carbon nanotubes–poly(methylene green) composite

A reagentless d-sorbitol biosensor based on NAD-dependent d-sorbitol dehydrogenase (DSDH) immobilized in a sol–gel carbon nanotubes–poly(methylene green) composite has been developed. It was prepared by durably immobilizing the NAD⁺ cofactor with DSDH in a sol–gel thin film on the surface of carbon nanotubes functionalized with poly(methylene green). This device enables selective determination of d-sorbitol at 0.2 V with a sensitivity of 8.7?μA?mmol^?1?L?cm^?2 and a detection limit of 0.11 mmol?L^?1. Moreover, this biosensor has excellent operational stability upon continuous use in hydrodynamic conditions.

Comparison of an HPTLC method with the Reflectoquant assay for rapid determination of 5-hydroxymethylfurfural in honey

5-Hydroxymethylfurfural (HMF) was analyzed in 17 botanical varieties of honey from 12 countries. A recently developed high-performance thin-layer chromatographic (HPTLC) method was limited because of increased matrix effects at higher honey sample loading. Therefore, the method was modified to achieve higher sensitivity and eliminate matrix interference by use of rectangular application combined with a focusing step. The HPTLC results were compared with results from the new spectrophotometric Reflectoquant hydroxymethylfurfural assay. Both methods had quantification limits of 4 mg kg^?1 and were suitable for rapid quantification of HMF in honey at the strictest regulated level of 15 mg kg^?1. Comparable results were obtained for the 17 honey samples, with a mean deviation of 2.9 mg kg^?1 (15 %). The optimized HPTLC method was proved to be highly matrix-robust and was validated for the 17 different honey matrices (correlation coefficients ≥0.9994 (n?=?6), mean intra-day precision 3.2 % (n?=?3 within a plate; n?=?2 repeated within a day), mean inter-day precision 3.7 % (n?=?3), mean reproducibility over the whole procedure including sample preparation 4.1 % (n?=?2), and mean recovery 106.9 % (n?=?5 different concentrations; n?=?4 different honey matrices). Recovery for a range of different application volumes, and thus for different honey matrix loading, differed by only ≤4.2 %. HMF results when calculated by use of external calibration and by use of the standard addition method varied by 8.8 %. Both revealed that any matrix effect was minor and that the original matrix interference problem was successfully solved.

Molecularly imprinted solid-phase extraction monolithic capillary column for selective extraction and sensitive determination of safranine T in wolfberry

A method was developed to sensitively determine safranine T in wolfberry by molecularly imprinted solid-phase extraction (MISPE) coupled with high-performance liquid chromatography and laser-induced fluorescence detection (HPLC-LIF). The MISPE capillary monolithic column was prepared by water-bath in situ polymerization, using safranine T, methacrylic acid (MAA), and ethylene dimethacrylate (EDMA) as template, functional monomer, and cross-linker, respectively. The properties of the homemade MISPE capillary monolithic column, including capacity and specificity, were investigated under optimized conditions and the morphologies of inner polymers were characterized by scanning electron microscopy (SEM). The mean recoveries of safranine T in wolfberry ranged from 91.2 % to 92.9 % and the intraday and interday relative standard deviation (RSD) values all ranged from 3.4 % to 4.2 %. Good linearity was obtained over 0.001–1.0 μg mL^–1 (r?=?0.9999) with a detection limit (S/N?=?3) of 0.4 ng g^–1. Under the selected conditions, enrichment factors of over 90-fold were obtained and the extraction on the monolithic column effectively cleaned up the wolfberry matrix. The results demonstrated that the proposed MISPE-HPLC-LIF method could be applied to sensitively determine safranine T in wolfberry.

Centri-voltammetric determination of glutathione

Centri-voltammetry is a method for concentrating an analyte on an electrode with the aid of a centrifuge. It can be performed in the presence or the absence of a solid carrier/support. This is followed by a voltammetric (analytical) scan. Specifically, we describe here an application of the technique to the determination of glutathione (GSH). Silica gel is used as the carrier precipitate to which gold nanoparticles were added in order to improve accumulation as a result of their strong affinity for thiols. Voltammetry was performed with a carbon paste electrode modified with multi-wall carbon nanotubes. The response to GSH is linear in the 25 and 800 μM concentration range (the correlation coefficient being 0.9915) and the relative standard deviation is 3.40 % (at 250 μM of GSH and n?=?6). The procedure was successfully applied to the determination of GSH in wine and in synthetic plasma using the standard addition method. The recoveries are 100.8 % and 100.0 %, respectively.

Development and validation method for the determination of selected tetracyclines in animal medicated feedingstuffs with the use of micellar liquid chromatography

A chromatographic procedure for the determination of oxytetracycline (OTC), tetracycline (TC), chlorotetracycline (CTC), and doxycycline (DC) in medicated feedingstuffs was developed. Samples were extracted with 0.01 M citric buffer/acetonitrile (pH 3.0) and further purified with 0.45 μm syringe filters. The purified extract was separated on Thermo column C₁₈, 150?×?4 mm, 5 μm and detection was carried out at 360 nm for OTC, and TC, 370 nm for CTC, and 350 nm for DC. TCs were eluted with a mobile phase of 0.03 M SDS/7 % 1-butanol/0.02 M oxalic acid/NaOH at pH 2.5. This method provided average recoveries of 80.4 % to 100.2 %, with CVs of 0.5 % to 6.6 % in the range of 50 to 1500 mg/kg OTC, TC, CTC, and DC in feeds. The linearity for the four TCs was determined by high-performance liquid chromatography-diode array detector (HPLC-DAD) in the range 10–300 μg/mL (50–1500 mg/kg), with a linear correlation coefficient (R)?>?0.99. The LOD and LOQ for TCs in pig and poultry feeds ranged from 4.0 to 10.7 and 4.7 to 12.6 mg/kg, respectively. The methodology was applied to the analysis of animal feedingstuffs collected from poultry and pig farms.

A silica-dextran nanocomposite as a novel matrix for immobilization of horseradish peroxidase, and its application to sensing hydrogen peroxide

We report on a novel matrix of solgel organic–inorganic nanocomposite that was fabricated from silica sol gel and dextran. It was used for the immobilization of horseradish peroxidase (HRP) to give a biosensor for hydrogen peroxide (H₂O₂). The sensor film was characterized by Fourier transform infrared and UV–vis spectroscopy with respect to structural features and the conformation of the enzyme. The topographies of the surface of the electrode were investigated by field emission scanning electron microscopy. The biosensor was used to determine H₂O₂ quantitatively in the presence of Methylene blue as a mediator with high electron transfer efficiency. A pair of stable and well defined quasi-reversible redox peaks of the HRP [Fe (III)]/HRP [Fe (II)] redox couple was observed at pH 7.0. The biosensor responds to H₂O₂ in the 0.5 mM to 16.5 mM concentration range, and the limit of detection is 0.5 mM.

Selective solid-phase extraction of trace mercury(II) using a silica gel modified with diethylenetriamine and thiourea

We describe a solid phase extractor for selective separation and preconcentration of Hg(II) ion. It was prepared by immobilizing the adduct of diethylenetriamine and thiourea on silica gel. The effects of solution acidity, preconcentration time, sample flow rate and volume were optimized. The results show that Hg(II) can be selectively extracted from acidic solutions and in presence of common other metal ions. The adsorbent is stable, can be reused more than 10 times, and the maximum adsorption capacity is 23 mg g^?1. Hg(II) was quantified by inductively coupled plasma optical emission spectrometry. The method has a detection limit of 23 ng L^?1, and the relative standard deviation is <2 %. The procedure was validated by analyzing two standard materials (river sediment and hair powder), and was successfully applied to the preconcentration of Hg(II) in real samples.

Preparation of chlorocholine chloride/urea deep eutectic solvent-modified silica and an examination of the ion exchange properties of modified silica as a Lewis adduct

Chlorocholine chloride/urea (ClChCl–urea), a deep eutectic solvent (DES), was applied successfully to the modification of silica. The resulting modified silica was characterized by Fourier transform infrared spectroscopy, Brunauer–Emmett–Teller analysis, and elemental analysis. Based on the ClChCl–urea modification of silica, the ClChCl–urea-modified silica is a Lewis adduct with ion exchange properties, and ferulic acid was adsorbed on the ClChCl–urea-modified silica via an ion exchange process. The adsorbed percentage of ferulic acid increased with the increasing amount of modified silica, and a high adsorbed percentage of 89 % could be obtained by the ion exchange process. The Freundlich isotherm used to describe the adsorption of ferulic acid on the modified silica by ion exchange showed a good correlation (R ²?=?0.93). Based on the characterization of the structure and the analysis of the ion exchange property of the ClChCl–urea-modified silica, the modified silica as a potential medium can be applied in some analytical technologies such as solid phase extraction, chromatography, and so on.

Preparation of ionic liquid/silica nanocomposites possessing no weight loss characteristic after calcination at 800 °C

Tetraethoxysilane reacted with silica nanoparticles in the presence of a variety of ionic liquids such as phosphonium-, ammonium-, and imidazolium-type ionic liquids under alkaline conditions to afford the corresponding ionic liquid/silica nanocomposites with a good dispersibility and stability in numerous solvents. It was demonstrated that these ionic liquid nanocomposites can be classified into the nanocomposites possessing no weight loss and weight loss characteristics after calcination at 800 °C, respectively, according to the ionic liquid structures in the composites. The ionic liquids except for 1-ethyl-3-methylimidazolium hydrogen sulfate were found to afford the corresponding ionic liquid/silica nanocomposites possessing no weight loss characteristic even after calcination at 800 °C.

Preparation and proton conductivity evaluation of a silicate gel composite doped with a metal–Schiff-base–POM-MOF

A silica gel composite (denoted as 1–SG) doped with a proton-conductive metal–Schiff-base–POM-MOF, {[Cu₃(L)₂(H₂O)₄][Cu(DMF)₄(SiW₁₂O₄₀)]·9H₂O} _n (1) (where L is N, N′-bis[1-(2-methoxyphenol-6-yl)-methylidene] hydrazine hydrate, DMF is dimethyl formamide, POM-MOF is polyoxometalates-based metal–organic framework), was prepared by sol–gel method. The structure of as-synthesized 1–SG was confirmed by infrared spectrometry and X-ray powder diffraction, and its proton conductivity was calculated based on electrochemical impedance spectroscopic measurement. It was found that the structural characteristics of complex 1 are retained successfully in the silica gel skeleton of as-prepared 1–SG. Besides, though 1–SG contains just 6.25 wt% complex 1, it exhibits good proton conductivities of as much as 1.51 × 10^?3–1.26 × 10^?2 S cm^?1 in the temperature range of 25–100 °C under a relative humidity of 98 %; and in particular, it shows better proton conductivity than both complex 1 and silica gel at the same conditions, due to the presence of a large number of micropores and mesopores filled with “liquid”.     

Poly(ethylene oxide)–poly(ethylene imine) block copolymers as templates and catalysts for the in situ formation of monodisperse silica nanospheres

Block copolymers based on poly(ethylene oxide) (PEO) and poly(ethylene imine) (PEI) are efficient catalysts/templates for the formation of uniform silica nanoparticles. Addition of tetraethylorthosilicate to a solution of PEO–PEI or PEI–PEO–PEI block copolymers results in the formation of silica particles with a diameter of ca. 30 nm and narrow size distribution. The particles precipitated with the diblock copolymers can be redispersed in water after isolation as individual nanoparticles. Evidently, block copolymers based on PEO and PEI serve as excellent templates for the biomimetic and “soft” synthesis of silica nanoparticles.

Preparation and characterization of film-forming raspberry-like polymer/silica nanocomposites via soap-free emulsion polymerization and the sol–gel process

Herein, we report on the synthesis of film-forming poly(styrene-co-butyl acrylate-co-acrylic acid)/SiO₂ [P(St-BA-AA)/SiO₂] nanocomposites by in situ formation of SiO₂ nanoparticles from TEOS via sol–gel process in the presence of poly(acrylic acid) (PAA)-functionalized poly(styrene-co-butyl acrylate) [P(St-BA)] particles fabricated by soap-free emulsion polymerization. The formed silica particles could be absorbed by polyacrylate chains on the surface of PAA-functionalized P(St-BA) particles; thus, raspberry-like polymer/silica nanocomposites would be obtained. Transmission electron microscopy, Fourier transform infrared spectroscopy, attenuated total reflectance infrared spectrum, ultraviolet–visible transmittance spectra, and thermogravimetric analysis were used to characterize the resulting composites. The results showed that the hybrid polymer/silica had a raspberry-like structure with silica nanoparticles anchored on the surface of polymer microspheres. The thermal, fire retardant, and mechanical properties and water resistance of the film were improved by incorporating silica nanoparticles, while the optical transmittance was seldom affected due to nanosized silica particles uniformly dispersed in the film.

Syntheses, crystal structures and fluorescent properties of two d 10-metal coordination polymers based on 1,5-naphthalenedisulfonic acid

The first examples of two d ¹⁰ metal coordination polymers based on 1,5-naphthalenedisulfonic acid and benzimidazole, namely, [Cd(bim)₄(1,5-nds)] 1 and [Ag(bim)₂]·½1,5-nds 2 (bim = benzimidazole, 1,5-nds = 1,5-naphthalenedisulfonic acid) were synthesized successfully under solvothermal conditions. Their structures were determined by single-crystal X-ray diffraction analyses and further characterized by elemental analyses, IR, TG-DSC, PXRD, UV–Vis, and DFT. 1 exhibits a 3D supramolecular structure which started from 1D helical chain. 2 also shows a 3D architecture which was assembled via π–π interactions and hydrogen bonds. The hydrogen bonds formed by 1,5-nds play an important role in increasing the dimensionality of architectures of 1 (from 1D to 3D) and 2 (from 0D to 1D). Moreover, both 1 and 2 show excellent luminescent properties, 335 nm for 1 (λ _ex = 286 nm) and 341 nm and 389 nm for 2 (λ _ex = 234 nm), and may be the suitable candidates of fluorescent materials. From UV–Vis spectra, the remarkable absorption peaks can be found at 276 nm for 1 and 271 nm for 2. The simulated UV–Vis spectra (290.30 nm for 1 and 271.64 nm for 2) by Gaussian 09 DFT correspond well with the experimental results. From the electron density distribution of frontier molecular orbitals, it is known that the two absorption bands are attributed to the intra-ligand electron transition from HOMO?26 to LUMO and from HOMO?3 to LUMO+1 for 1, from HOMO?1 to LUMO+18 and LUMO+19 for 2.     

UPLC-UV-MSE analysis for quantification and identification of major carotenoid and chlorophyll species in algae

A fast method for quantification and identification of carotenoid and chlorophyll species utilizing liquid chromatography coupled with UV detection and mass spectrometry has been demonstrated and validated for the analysis of algae samples. This method allows quantification of targeted pigments and identification of unexpected compounds, providing isomers separation, UV detection, accurate mass measurements, and study of fragment ions for structural elucidation in a single run. This is possible using parallel alternating low- and high-energy collision spectral acquisition modes, which provide accurate mass full scan chromatograms and accurate mass high-energy chromatograms. Here, it is shown how this approach can be used to confirm carotenoid and chlorophyll species by identification of key diagnostic fragmentations during high-energy mode. The developed method was successfully applied for the analysis of Dunaliella salina samples during defined red LED lighting growth conditions, identifying 37 pigments including 19 carotenoid species and 18 chlorophyll species, and providing quantification of 7 targeted compounds. Limit of detections for targeted pigments ranged from 0.01?ng/mL for lutein to 0.24?ng/mL for chlorophyll a. Inter-run precision ranged for of 3 to 24 (RSD%) while inter-run inaccuracy ranged from ?17 to 11.

Analysis of Biomolecules by Atmospheric Pressure Visible-Wavelength MALDI-Ion Trap-MS in Transmission Geometry

We report the development of a new AP visible-wavelength MALDI-ion trap-MS instrument with significantly improved performance over our previously reported system (Int. J. Mass Spectrom. 315, 66–73 (2012)). A Nd:YAG pulsed laser emitting light at 532 nm was used to desorb and ionize oligosaccharides and peptides in transmission geometry through a glass slide. Limits of detection (LODs) achieved in MS mode correspond to picomole quantities of oligosaccharides and femtomole quantities of peptides. Tandem MS (MS/MS) experiments enabled identification of enzymatically digested proteins and oligosaccharides by comparison of MS/MS spectra with data found in protein and glycan databases. Moreover, the softness of ionization, LODs, and fragmentation spectra of biomolecules by AP visible-wavelength MALDI-MS were compared to those obtained by AP UV MALDI-MS using a Nd:YAG laser emitting light at 355 nm. AP visible-wavelength MALDI appears to be a softer ionization technique then AP UV MALDI for the analysis of sulfated peptides, while visible-wavelength MALDI-MS, MS/MS, and MS/MS/MS spectra of other biomolecules analyzed were mostly similar to those obtained by AP UV MALDI-MS. Therefore, the methodology presented will be useful for MS and MSⁿ analyses of biomolecules at atmospheric pressure. Additionally, the AP visible-wavelength MALDI developed can be readily used for soft ionization of analytes on various mass spectrometers.

Silica-coated magnetic nanoparticles loaded with Cu(II): preparation and application to the purification of bovine serum albumin

Magnetic nanoparticles (MNPs) coated with silica gel were prepared, then functionalized with a tridentate ligand via a silane coupling agent (3-chloropropyl)triethoxysilane, and finally loaded with Cu(II) ions. The resulting materials were characterized by TEM, SEM, XRD, FTIR and TGA techniques. They display strong affinity for BSA with an adsorption capacity as high as 235 mg g^?1 and with a fast (30 min) establishment of adsorption equilibrium. Repetitive adsorptions (6 times) hardly affect the adsorption capability. The kinetics and isotherm of the adsorption of BSA were also investigated.