We characterized thermally polymerized organo-silica hybrid monolithic capillaries to test their applicability in the gradient elution of peptides. We have used a single-pot approach utilizing 3-(methacryloyloxy)propyltrimethoxysilane (MPTMS), ethylene dimethacrylate (EDMA), and n-octadecyl methacrylate (ODM) as functional monomers. The organo-silica monolith containing MPTMS and EDMA was compared with the stationary phase prepared by adding ODM to the original polymerization mixture. Column prepared using a three-monomer system provided a lower accessible volume of flow-through pores, a higher proportion of mesopores, and higher efficiency. We utilized isocratic and gradient elution data to predict peak widths in gradient elution. Both protocols provided comparable results and can be used for peptide peak width prediction. However, applying gradient elution data for peak width prediction seems simpler. Finally, we tested the effect of gradient time on achievable peak capacity in the gradient elution of peptides with a column prepared with a three-monomer system providing a higher peak capacity. However, the performance of hybrid organo-silica monolithic stationary phases in gradient elution of peptides must be improved compared to other monolithic stationary phases. The limiting factor is column efficiency in highly aqueous mobile phases, which needs to be focused on.

• Klíčová slova
• gradient elution, hybrid organo-silica monoliths, kinetic analysis, peptides,
• MeSH
• methakryláty chemie   MeSH
• oxid křemičitý * chemie   MeSH
• peptidy * chemie   MeSH
• voda MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ethylene dimethacrylate MeSH Prohlížeč
• methakryláty MeSH
• oxid křemičitý * MeSH
• peptidy * MeSH
• voda MeSH

Gradient elution is widely used for separation of complex samples in reversed-phase HPLC systems, but is less frequently applied in normal-phase HPLC, where it has a notoriously bad reputation for poor reproducibility and unpredictable retention. This behaviour is caused by preferential adsorption of polar solvents used in mixed mobile phases, which may cause significant deviations of the actual gradient profile from the pre-set program. Another important source of irreproducible retention behaviour is gradual deactivation of the adsorbent by adsorption of even traces of water during normal-phase gradient elution. To avoid this phenomenon, carefully dried solvents should be used. Finally, column temperature should be carefully controlled during normal-phase gradient elution if reproducible results are to be obtained. Working with dry solvents at a controlled constant temperature and using a sophisticated gradient-elution chromatograph, reproducibility of the retention data in normal-phase gradient elution better than 2% may be achieved even over several months of column use. The retention data in gradient elution can be calculated accurately if appropriate corrections are adopted for the gradient dwell volume and for the preferential adsorption of the polar solvents using experimental adsorption isotherms. The average error of prediction for the corrected calculated gradient retention data was lower than 2% for a silica gel column and lower than 3% for a bonded nitrile column, which may be suitable for the optimization of separation. Further, a simple approach is suggested for rapid estimation of changes in the retention induced by a change in the gradient profile in normal-phase HPLC. For such a rough estimation, it is not necessary to know the parameters of the dependence of the solute retention factors on the composition of the mobile phase.

• MeSH
• pesticidy analýza   MeSH
• spektrofotometrie ultrafialová MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• pesticidy MeSH

The paper deals with the concept of simple automated creation of gradient profile of the mobile phase for gradient-elution sequential injection chromatography (GE-SIC). The feasibility and merits of this concept are demonstrated on the separation and simultaneous assay of indomethacin as active principle and of its two degradation products (5-methoxy-2-methylindoleacetic acid and 4-chloro-benzoic acid) in a topical pharmaceutical formulation. The GE-SIC separation was performed with a FIAlab(®) 3000 SIC set-up (USA) equipped with an Onyx™ Monolithic C18 (25 mm × 4.6mm, Phenomenex(®)) column, a six-port selection valve, a 5-mL syringe pump and a fiber-optics UV CCD detector. Ketoprofen was used as an internal standard (IS). The gradient elution was achieved by automated reproducible mixing of acetonitrile and aqueous 0.2% phosphoric acid in the holding coil of the SIC system. Different profiles of the gradient elution were tested. The optimal gradient using two mobile phases 30:70 and 50:50 of acetonitrile/0.2% phosphoric acid (v/v) was achieved under the optimum flow rate 1.2 mL min(-1). The chromatographic resolution R between the peaks of all solutes (including the IS) was >2.00. The repeatability of retention times was characterized by the RSD values 0.18-0.30% (n=6). Net separation time was 3.5 min and the mobile phase consumption was 4.5 mL for a single GE-SIC assay. The figures of merit of the novel GE-SIC method compared well with those of conventional HPLC.

• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

To investigate the effects of various parameters on the production rate and on the recovery yield in overloaded reversed-phase gradient-elution chromatography, band profiles of binary mixtures of phenol and o-cresol were calculated using the experimental parameters of the distribution isotherms determined by binary frontal analysis. The effects of the feed volume and of the steepness of a continuous gradient (gradient time) of methanol in aqueous-organic mobile phases on the separation were studied. If the sample feed in a solvent with weak elution strength is used, combined effects of on-column enrichment, frontal chromatography and sharpening of the later eluted bands may enhance the production rate and the recovery yield. Steep continuous gradients offer better results than isocratic elution if the feed is dissolved in water and injected into the mobile phase with a higher elution strength, because larger volumes of dilute feed can be separated with high recovery yields.

• MeSH
• chemické modely MeSH
• chromatografie kapalinová metody   MeSH
• fenol chemie   MeSH
• kresoly chemie   MeSH
• osmolární koncentrace MeSH
• reprodukovatelnost výsledků MeSH
• spektrofotometrie ultrafialová MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• srovnávací studie MeSH
• Názvy látek
• 2-cresol MeSH Prohlížeč
• fenol MeSH
• kresoly MeSH

The possibility of applying a theoretical model in the prediction of the retention of phenolic acids on hydrosilated silica, in aqueous normal phase mode was studied. The actual gradient of the aqueous component in acetonitrile may fluctuate from the pre-set program, as even the gradient-grade acetonitrile contains some water. Hence, the actual concentration of water during the gradient run is higher than pre-set by the gradient program, which leads to lower than expected sample retention. Furthermore, the actual gradient profile may be affected by an increase in water uptake on a polar column during the gradient run. These effects were investigated using the using frontal analysis method and Karl-Fischer titration, for the determination of water in the initial mobile phase, and in the column effluent. Preferential adsorption of water on the Silica hydride, Diamond hydride, UDC Cholesterol, Bidentate C18, and Phenyl hydride columns can be described by Langmuir isotherms. At the column saturation capacity, less than one monomolecular water layer is adsorbed, with a further decrease in coverage density for modified materials. Parameters of semi-logarithmic and logarithmic model equations, describing the dependence of retention factor on the concentration of water, were determined under isocratic conditions. These parameters and linear gradient profiles corrected for the actual water concentrations were used in calculation of gradient retention data. The corrections for the actual water concentration greatly improved the agreement between the experiment and the predicted gradient elution volumes. Generally, the semi-logarithmic model provides slightly better prediction of the gradient data, with respect to the logarithmic retention model.

• MeSH
• adsorpce MeSH
• chromatografie kapalinová přístrojové vybavení   metody   MeSH
• hydroxybenzoáty chemie   MeSH
• oxid křemičitý chemie   MeSH
• voda chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hydroxybenzoáty MeSH
• oxid křemičitý MeSH
• phenolic acid MeSH Prohlížeč
• voda MeSH

Retention prediction of monoamine neurotransmitters has been compared for the generally applied linear solvent-strength model and quadratic polynomial three-parameter model. The design of experiments protocol has been applied to plan linear gradients within the experimental space with altered gradient time, mobile phase flow rate, and column temperature. Relative prediction errors increased at elevated temperature, which is more significant for the linear solvent-strength model when compared to the polynomial model. On the other hand, the predefined design of experiments space controls the retention time errors, as predictions for LC conditions that are outside of the plan are much less accurate and should be avoided. The final part of the work deals with the effect of extracolumn band dispersion on the peak capacity of linear gradients at various gradient times, mobile phase flow rates, and column temperature. The peak capacity determined for corrected experimental data were consistent with the published results dealing with the optimization of peak capacity in gradient elution.

• Klíčová slova
• design of experiments, extracolumn volume, gradient elution, peak capacity, retention modeling,
• MeSH
• chromatografie kapalinová metody   MeSH
• indikátory a reagencie MeSH
• lineární modely MeSH
• neurotransmiterové látky * MeSH
• rozpouštědla chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• indikátory a reagencie MeSH
• neurotransmiterové látky * MeSH
• rozpouštědla MeSH

This work presents novel approach in low-pressure chromatography flow systems--two-column Sequential Injection Chromatography (2-C SIC) and its comparison with gradient elution chromatography on the same instrument. The system was equipped with two different chromatographic columns (connected to selection valve in parallel design) for isocratic separation and determination of all components in composed anti-inflammatory pharmaceutical preparation (tablets). The sample was first injected on the first column of length 30 mm where less retained analytes were separated and then the sample was injected on the second column of length 10 mm where more retained analytes were separated. The SIC system was based on a commercial SIChrom manifold (8-port high-pressure selection valve and medium-pressure syringe pump with 4 mL reservoir) (FIAlab, USA) with two commercially available monolithic columns the "first column" Chromolith Flash RP-18e (25 mm x 4.6 mm i.d. with guard column 5 mm x 4.6 mm i.d.) and the "second column" Chromolith RP-18e (10 mm x 4.6 mm i.d.) and CCD UV-vis detector USB 4000 with micro-volume 1.0 cm Z flow cell. Two mobile phases were used for analysis (one for each column). The mobile phase 1 used for elution of paracetamol, caffeine and salicylic acid (internal standard) was acetonitrile/water (10:90, v/v, the water part of pH 3.5 adjusted with acetic acid), flow rate was 0.9 mL min(-1) (volume 3.0 mL of mobile phase per analysis). The mobile phase 2 used for elution of propyphenazone was acetonitrile/water (30:70, v/v); flow rate was 1.2 mL min(-1) (volume 1.5 mL of mobile phase per analysis). Absorbance was monitored at 210 nm. Samples were prepared by dissolving of one tablet in 30% acetonitrile and 10 microL of filtered supernatant was injected on each column (2 x 10 microL). The chromatographic resolution between all compounds was >1.45 and analysis time was 5.5 min under the optimal conditions. Limits of detection were determined at 0.4 microg mL(-1) for paracetamol, at 0.5 microg mL(-1) for caffeine and at 0.7 microg mL(-1) for propyphenazone. The new two-column chromatographic set-up developed as an alternative approach to gradient elution chromatography shows evident advantages (time and solvent reduction more than one-third) as compared with single-column gradient SIC method with Chromolith Flash RP-18 (25 mm x 4.6 mm i.d. with guard column 5 mm x 4.6 mm i.d.).

• MeSH
• antiflogistika nesteroidní analýza   chemie   MeSH
• fenazon analogy a deriváty   analýza   MeSH
• kofein analýza   MeSH
• paracetamol analýza   MeSH
• spektrofotometrie ultrafialová MeSH
• tablety chemie   MeSH
• vysokoúčinná kapalinová chromatografie přístrojové vybavení   metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• antiflogistika nesteroidní MeSH
• fenazon MeSH
• kofein MeSH
• paracetamol MeSH
• propyphenazone MeSH Prohlížeč
• tablety MeSH

The most active form of sulfur in biomolecules is the thiol group, present in a number of biologically active compounds. Here we present a comprehensive study of thiol analysis using flow injection analysis/HPLC with electrochemical detection. The effect of different potentials of working electrodes, of organic solvent contents in the mobile phase, and of isocratic and gradient elution on simultaneous determination of thiol compounds (cysteine, cystine, N-acetylcysteine, homocysteine, reduced and oxidised glutathione, desglycinephytochelatin, and phytochelatins) are described and discussed. These thiol compounds were well separated and detected under optimised HPLC-electrochemical detection conditions (mobile phase: 80 mM trifluoroacetic acid and methanol with a gradient profile starting at 97:3 (TFA:methanol), kept constant for the first 8 min, then decreasing to 85:15 during one minute, kept constant for 8 min, and finally increasing linearly up to 97:3 from 17 to 18 min; the flow rate was 0.8 mL/min, column and detector temperature 25 degrees C, and the electrode potential 900 mV). We were able to determine tens of femtomoles (3 S/N) of the thiols per injection (5 microL), except for phytochelatin5 whose detection limit was 2.1 pmole. This technique was consequently used for simultaneous determination of compounds of interest in biological samples (maize tissue and human blood serum).

• MeSH
• chromatografie kapalinová * přístrojové vybavení   metody   MeSH
• kukuřice setá chemie   MeSH
• lidé MeSH
• molekulární struktura MeSH
• reprodukovatelnost výsledků MeSH
• rozpouštědla chemie   MeSH
• senzitivita a specificita MeSH
• sérum chemie   MeSH
• sulfhydrylové sloučeniny analýza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• rozpouštědla MeSH
• sulfhydrylové sloučeniny 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

Performing gradient liquid chromatography at constant pressure instead of constant flow rate has serious potential for shortening the analysis time and increasing the productivity of HPLC instruments that use gradient methods. However, in the constant pressure mode the decreasing column permeability during a long period of time negatively affects the repeatability of retention time. Thus a volume-based approach, in which the detector signal is plotted as a function of retention volume, must be taken into consideration. Traditional HPLC equipment, however, requires quite complex hardware and software modifications in order to work at constant pressure and in the volume-based mode. In this short communication, a low cost and easily feasible pressure-controlled extension of the previously described simple gradient liquid chromatography platform is proposed. A test mixture of four nitro esters was separated by 10-60% (v/v) acetone/water gradient and a high repeatability of retention volumes at 20MPa (RSD less than 0.45%) was realized. Separations were also performed at different values of pressure (20, 25, and 31MPa), and only small variations of the retention volumes (up to 0.8%) were observed. In this particular case, the gain in the analysis speed of 7% compared to the constant flow mode was realized at a constant pressure.

• Klíčová slova
• Constant pressure HPLC, Gradient elution, Nanocolumns, Nitro esters, Simple liquid chromatograph,
• MeSH
• chemie přístrojové vybavení   MeSH
• chromatografie kapalinová přístrojové vybavení   MeSH
• estery chemie   MeSH
• tlak * MeSH
• voda chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• estery MeSH
• voda MeSH