High-resolution monolith Dotaz Zobrazit nápovědu
The reduction of analysis time, cost, and improvement of separation efficiency are the main requirements in the development of high-throughput assay methods in bioanalysis. It can be achieved either by ultra-high-performance liquid chromatography (UHPLC) using stationary phases with small particles (<2 μm) at high back pressures or by using opposite direction--monolithic stationary phases with low back pressures. The application of new types of monolithic stationary phases for UHPLC is a novel idea combining these two different paths. The aim of this work was to test the recently introduced second-generation of monolithic column Chromolith® HighResolution for UHPLC analysis of liposoluble vitamins in comparison with core-shell and fully porous sub-2 μm columns with different particle sizes, column lengths, and shapes. The separation efficiency, peak shape, resolution, time of analysis, consumption of mobile phase, and lifetime of columns were calculated and compared. The main purpose of the study was to find a new, not only economical option of separation of liposoluble vitamins for routine practice.
- Klíčová slova
- Bioanalysis, Core-shell, High-resolution monolith, LC, Liposoluble vitamins,
- MeSH
- alfa-tokoferol analýza krev MeSH
- lidé MeSH
- mateřské mléko chemie MeSH
- poréznost MeSH
- syntetické pryskyřice chemie MeSH
- velikost částic MeSH
- vitamin A analýza krev MeSH
- vysokoúčinná kapalinová chromatografie přístrojové vybavení metody MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- alfa-tokoferol MeSH
- syntetické pryskyřice MeSH
- vitamin A MeSH
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 C(18), 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.
- MeSH
- adsorpce MeSH
- kofein chemie izolace a purifikace MeSH
- kyselina askorbová chemie izolace a purifikace MeSH
- paracetamol chemie izolace a purifikace MeSH
- vysokoúčinná kapalinová chromatografie přístrojové vybavení metody MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- Názvy látek
- kofein MeSH
- kyselina askorbová MeSH
- paracetamol MeSH
Two-dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On-line coupling of a reversed-phase column with an aqueous normal-phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on-line two-dimensional liquid chromatography needs a capillary or micro-bore column providing low-volume effluent fractions transferred to a short efficient second-dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro-columns in fused silica capillaries with structurally different dimethacrylate cross-linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed-phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on-line an organic polymer monolithic capillary column in the first dimension with a short silica-based monolithic column in the second dimension provides two-dimensional liquid chromatography systems with high peak capacities. The silica monolithic C18 columns provide higher separation efficiency than the particle-packed columns at the flow rates as high as 5 mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed-phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.
- Klíčová slova
- comprehensive two-dimensional chromatography, monolithic columns, polymethacrylate monoliths, silica-based monolithic columns,
- Publikační typ
- časopisecké články MeSH
We studied possibilities of prediction of the gradient elution data for alkylbenzenes, flavones and phenolic acids on two short octadecyl silica gel monolithic columns, namely a Chromolith Flash C18, 25×4.6mm, and a "new generation" Chromolith High Resolution C18, 50×4.6mm, in fast 1-2min gradients. With fixed short gradient times and varying gradient ranges of acetonitrile concentration in water, high flow rates of the mobile phase (3-5mL/min) could be used. The gradient elution data were predicted from four gradient models based on two-parameter and three-parameter isocratic retention equations. Various gradient retention models can be used for prediction of chromatograms and optimization of separation within a fixed gradient time. A two-parameter log-log model introduced in 1974 and a three-parameter model introduced in 1980 provided slightly more accurate prediction than the Linear Solvent Strength (LSS) semi-logarithmic two-parameter model, most frequently used in reversed-phase LC. A three-parameter model introduced in 1978 provided slightly improved accuracy of prediction of gradient data with respect to two-parameter models, in contrast to another, more recent three-parameter empirical model introduced in 2010 (which failed for gradients starting at a non-zero concentration of acetonitrile). Both a longer (5cm) and more efficient Chromolith HR column and a shorter (2.5cm) slightly less efficient Chromolith Flash column provide useful separations in fast gradients (1-2min) at high flow rates (3.5-5mL/min), especially in second dimension of two-dimensional LC×LC, in combination with HILIC separation on monolithic microcolumn in D1.
- Klíčová slova
- Fast separations, Gradient elution, Modeling of chromatography, Monolithic columns, Two-dimensional chromatography,
- MeSH
- acetonitrily MeSH
- benzenové deriváty analýza MeSH
- chromatografie kapalinová přístrojové vybavení metody MeSH
- flavony analýza MeSH
- oxid křemičitý chemie MeSH
- rozpouštědla MeSH
- statistické modely MeSH
- voda MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- acetonitrile MeSH Prohlížeč
- acetonitrily MeSH
- benzenové deriváty MeSH
- flavony MeSH
- octadecylsilica MeSH Prohlížeč
- oxid křemičitý MeSH
- rozpouštědla MeSH
- voda MeSH
A novel and fast simultaneous determination of triamcinolone acetonide (TCA) and salicylic acid (SA) in topical pharmaceutical formulations by sequential injection chromatography (SIC) as an alternative to classical high performance liquid chromatography (HPLC) has been developed. A recently introduced Onyxtrade mark monolithic C18 (50mmx4.6mm, Phenomenex((R))) with 5mm monolithic precolumn were used for the first time for creating sequential injection chromatography system based on a FIAlab((R)) 3000 with a six-port selection valve and 5.0mL syringe pump in study. The mobile phase used was acetonitrile/water (35:65, v/v), pH 3.3 adjusted with acetic acid at flow rate 0.9mLmin(-1). UV detection provided by fibre-optic DAD detector was set up at 240nm. Propylparaben was chosen as suitable internal standard (IS). There is only simple pre-adjustment of the sample of topical solution (dilution with mobile phase) so the analysis is not uselessly elongated. Parameters of the method showed good linearity in wide range, correlation coefficient >0.999; system precision (relative standard deviation, R.S.D.) in the range 0.45-1.95% at three different concentration levels, detection limits (3sigma) 1.00mugmL(-1) (salicylic acid), 0.66mugmL(-1) (triamcinolone acetonide) and 0.33mugmL(-1) (propylparaben) and recovery from the pharmaceutical preparations in the range 97.50-98.94%. The chromatographic resolution between peaks of compounds was more than 4.5 and analysis time was 5.1min under the optimal conditions. The advantages of sequential injection chromatography against classical HPLC are discussed and showing that SIC can be a method of option in many cases.
- Publikační typ
- časopisecké články MeSH
AIM: A new HPLC method for the determination of neopterin, kynurenine and tryptophan using a second-generation monolith stationary phase and high-throughput sample preparation procedure based on microplates was developed and fully validated. MATERIALS & METHODS: As the stationary phase a monolithic C18 Chromolith high-resolution column with dimensions of 4.6 × 100 mm connected to a monolithic 4.6 × 10-mm security guard was used. Separation was achieved using 15 mM phosphate buffer (KH2PO4 +K2HPO4·3H2O at pH 3) and acetonitrile in gradient mode. RESULTS: Target analytes were determined in 5.5 minutes in amniotic fluid, effusions and wound exudates with a limit of quantification (LOQ) of 1.25 nM for neopterin, 2.5 µM for tryptophan and 0.25 µM for kynurenine. DISCUSSION: The method was applied to real clinical sample measurements, and it will be used to monitor neopterin, kynurenine and tryptophan levels in biological fluids to assess the patient response to therapy and clinical status.
- MeSH
- exsudáty a transsudáty metabolismus MeSH
- hojení ran * MeSH
- kynurenin analýza MeSH
- lidé MeSH
- maligní pleurální výpotek metabolismus MeSH
- neopterin analýza MeSH
- plodová voda metabolismus MeSH
- spektrofotometrie ultrafialová MeSH
- tryptofan analýza MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- kynurenin MeSH
- neopterin MeSH
- tryptofan MeSH
Biomaterial for tissue engineering is a topic of huge progress with a recent surge in fabrication and characterization advances. Biomaterials for tissue engineering applications or as scaffolds depend on various parameters such as fabrication technology, porosity, pore size, mechanical strength, and surface available for cell attachment. To serve the function of the scaffold, the porous biomaterial should have enough mechanical strength to aid in tissue engineering. With a new manufacturing technology, we have obtained high strength materials by optimizing a few processing parameters such as pressure, temperature, and dwell time, yielding the monolith with porosity in the range of 80%⁻93%. The three-dimensional interconnectivity of the porous media through scales for the newly manufactured biomaterial has been investigated using newly developed 3D correlative and multi-modal imaging techniques. Multiscale X-ray tomography, FIB-SEM Slice & View stacking, and high-resolution STEM-EDS electronic tomography observations have been combined allowing quantification of morphological and geometrical spatial distributions of the multiscale porous network through length scales spanning from tens of microns to less than a nanometer. The spatial distribution of the wall thickness has also been investigated and its possible relationship with pore connectivity and size distribution has been studied.
- Klíčová slova
- biomaterials, bone regeneration, microstructure, porous materials, tissue engineering,
- Publikační typ
- časopisecké články MeSH
Any bio-analytical method includes several steps, all of them being important in order to achieve reliable results. The first step is taking aliquots of samples for the analysis, followed by the extraction procedure and sample clean-up, chromatographic analysis and detection. Chromatographic methods, particularly liquid chromatography, are the methods of choice in bio-analytical laboratories. Current trends in fast liquid chromatographic separations involve monolith technology, fused core columns, high temperature liquid chromatography and ultra-high performance liquid chromatography (UHPLC). UHPLC has recently become a wide-spread analytical technique in many laboratories which focus on fast and sensitive bio-analytical assays. The key advantages of UHPLC are the increased speed of analysis, higher separation efficiency and resolution, higher sensitivity and much lower solvent consumption as compared to other analytical approaches. This is all enabled by specially designed instruments and sub-2-microne particle packed analytical columns. There is a great contrast between ultra-fast chromatographic analysis and conventional sample preparation, which remains highly labor-intensive and time-consuming. Conventional sample preparation techniques including SPE, solid phase extraction; LLE, liquid-liquid extraction; PP, protein precipitation and many modern approaches (RAM, restricted access material; MIP, molecularly imprinted polymers; SPME, solid phase microextraction; LLME, liquid-liquid microextraction; MEPS, microextraction by packed sorbent and many others) have also been featured as fundamental and critical step of bio-analytical methods.
The selection of column packing during the development of high-performance liquid chromatography method is a crucial step to achieve sufficient chromatographic resolution of analyzed species in complex mixtures. Various stationary phases are tested in this paper for the analysis of complex mixture of triacylglycerols (TGs) in blackcurrant oil using non-aqueous reversed-phase (NARP) system with acetonitrile-2-propanol mobile phase. Conventional C(18) column in the total length of 45 cm is used for the separation of TGs according to their equivalent carbon number, the number and positions of double bonds and acyl chain lengths. The separation of TGs and their more polar hydrolysis products after the partial enzymatic hydrolysis of blackcurrant oil in one chromatographic run is achieved using conventional C(18) column. Retention times of TGs are reduced almost 10 times without the loss of the chromatographic resolution using ultra high-performance liquid chromatography with 1.7 microm C(18) particles. The separation in NARP system on C(30) column shows an unusual phenomenon, because the retention order of TGs changes depending on the column temperature, which is reported for the first time. The commercial monolithic column modified with C(18) is used for the fast analysis of TGs to increase the sample throughput but at cost of low resolution.
- MeSH
- hmotnostní spektrometrie metody MeSH
- hydrolýza MeSH
- kyselina gama-linolenová chemie metabolismus MeSH
- lipasa chemie metabolismus MeSH
- teplota MeSH
- triglyceridy analýza MeSH
- vysokoúčinná kapalinová chromatografie přístrojové vybavení metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- black currant oil MeSH Prohlížeč
- kyselina gama-linolenová MeSH
- lipasa MeSH
- Lipozyme MeSH Prohlížeč
- triglyceridy MeSH
A novel approach for automation of Micro-Extraction by Packed Sorbent (MEPS), a solid phase extraction technique, is presented, enabling precise and repeatable liquid handling due to the employment of sequential injection technique. The developed system was used for human urine sample clean-up and pre-concentration of betaxolol before its separation and determination. A commercial MEPS C-18 cartridge was integrated into an SIChrom™ system. The chromatographic separation was performed on a monolithic High Resolution C18 (50×4.6 mm) column which was coupled on-line in the system with Micro-Extraction using an additional selection valve. A mixture of acetonitrile and aqueous solution of 0.5% triethylamine with acetic acid, pH adjusted to 4.5 in ratio 30:70 was used as a mobile phase for elution of betaxolol from MEPS directly onto the monolithic column where the separation took place. Betaxolol was quantified by a fluorescence detector at wavelengths λ(ex)=220 nm and λ(em)=305 nm. The linear calibration range of 5-400 ng mL(-1), with limit of detection 1.5 ng mL(-1) and limit of quantification 5 ng mL(-1) and correlation r=0.9998 for both the standard and urine matrix calibration were achieved. The system recovery was 105±5%; 100±4%; 108±1% for three concentration levels of betaxolol in 10 times diluted urine - 5, 20 and 200 ng mL(-1), respectively.
- Klíčová slova
- Automation, Betaxolol, Fluorimetry, Liquid chromatography, Micro-Extraction by Packed Sorbent (MEPS), Sequential Injection Chromatography (SIC),
- MeSH
- analýza moči přístrojové vybavení metody MeSH
- betaxolol izolace a purifikace moč MeSH
- chromatografie metody MeSH
- design vybavení MeSH
- fluorescenční spektrometrie MeSH
- injekce * MeSH
- lidé MeSH
- mikroextrakce na pevné fázi metody MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- betaxolol MeSH
