Szabolcs Fekete

University of Lausanne, Lausanne, Vaud, Switzerland

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Publications (69)212.17 Total impact

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    Balázs Bobály · Davy Guillarme · Szabolcs Fekete
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    ABSTRACT: A new superficially porous material possessing a carbon core and nanodiamond-polymer shell and pore size of 180Å was evaluated for the analysis of large proteins. Because the stationary phase on this new support contains a certain amount of protonated amino groups within the shell structure, the resulting retention mechanism is most probably a mix between reversed phase and anion exchange. However, under the applied conditions (0.1-0.5% TFA in the mobile phase), it seemed that the main retention mechanism for proteins was hydrophobic interaction with the C18 alkylchains on this carbon based material. In this study, we demonstrated that there was no need to increase mobile phase temperature, as the peak capacity was not modified considerably between 30 and 80°C for model proteins. Thus, the risk of thermal on-column degradation or denaturation of large proteins is not relevant. Another important difference compared to silica-based materials is that this carbon-based column requires larger amount of TFA, comprised between 0.2 and 0.5%. Finally, it is important to mention that selectivity between closely related proteins (oxidized, native and reduced forms of Interferon α-2A variants) could be changed mostly through mobile phase temperature. Copyright © 2014 Elsevier B.V. All rights reserved.
    Full-text · Article · Nov 2014 · Journal of Pharmaceutical and Biomedical Analysis
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    ABSTRACT: The goal of this study was to evaluate the combination of powerful chromatographic methods and compact single quadrupole MS device for simple in vitro cytochrome P450 (CYP) inhibition assay, instead of more expensive triple quadrupole MS/MS detectors. For this purpose, two modern chromatographic approaches (ultra-high pressure liquid chromatography (UHPLC) and ultra-high performance supercritical fluid chromatography (UHPSFC)) were tested in combination with simple MS detector. To show the applicability for an in vitro CYP-mediated metabolism assay using the cocktail approach, a method was first developed in UHPLC-MS to separate a mixture of 8 probe substrates and 8 CYP-specific metabolites. A screening procedure was initially applied to determine the best combination of a column, an organic modifier and a mobile-phase pH, followed by fine tuning of the conditions (i.e., gradient profile, temperature and pH) using HPLC modeling software. A similar sequential method development procedure was also evaluated for UHPSFC-MS. For method development where peak tracking is necessary, the use of single quadrupole MS was found to be extremely valuable for following the investigated analytes. Ultimately, a baseline separation of the 16 compounds was achieved in both UHPLC-MS and UHPSFC-MS with an analysis time of less than 7 minutes. In a second series of experiments, sensitivity was evaluated, and LOQ values were between 2 and 100 ng/mL in UHPLC-MS, while they ranged from 2 to 200 ng/mL in UHPSFC-MS. Based on the concentrations employed for the current in vitro phase I metabolism assay, these LOQ values were appropriate for this type of application. Finally, the two analytical methods were applied to in vitro CYP-dependent metabolism testing. Two well-known phytochemical inhibitors, yohimbine and resveratrol, were investigated, and reliable conclusions were drawn from these experiments with both UHPLC-MS and UHPSFC-MS. At the end, the proposed strategy of optimized chromatography combined with simple MS device has been shown to potentially compete with the widely used combination of generic chromatography and highly selective MS/MS device for simple in vitro CYP inhibition assays. In addition, our analytical method may be easier to use in a routine environment; the instrument cost is significantly reduced and the two developed method fit for purpose.
    Full-text · Article · Oct 2014 · Journal of Chromatography A
  • Szabolcs Fekete · Alain Beck · Jenő Fekete · Davy Guillarme
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    ABSTRACT: Ion exchange chromatography (IEX) is a historical technique widely used for the detailed characterization of therapeutic proteins and can be considered as a reference and powerful technique for the qualitative and quantitative evaluation of charge variants. When applying salt gradient IEX approach for monoclonal antibodies (mAbs) characterization, this approach is described as time-consuming to develop and product-specific. The goal of this study was to tackle these two bottle-necks. By modeling the retention of several commercial mAbs and their variants in IEX, we proved that the mobile phase temperature was not relevant for tuning selectivity, while optimal salt gradient program can be easily found based on only two initial gradients of different slopes. Last but not least, the dependence of retention vs. pH being polynomial, three initial runs at different pH were required to optimize mobile phase pH. Finally, only 9h of initial experiments were necessary to simultaneously optimize salt gradient profile and pH in IEX. The data can then be treated with commercial modeling software to find out the optimal conditions to be used, and accuracy of retention times prediction was excellent (less than 1% variation between predicted and experimental values). Second, we also proved that generic IEX conditions can be applied for the characterization of mAbs possessing a wide range of pI, from 6.7 to 9.1. For this purpose, a strong cation exchange column has to be employed at a pH below 6 and using a proportion of NaCl up to 0.2M. Under these conditions, all the mAbs were properly eluted from the column. Therefore, salt gradient CEX can be considered as a generic multi-product approach.
    No preview · Article · Sep 2014 · Journal of Pharmaceutical and Biomedical Analysis
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    ABSTRACT: This paper describes a new and fast ultra-high pressure liquid chromatographic separation of amlodipine and bisoprolol and all their closely related compounds, for impurity profiling purposes. Computer-assisted method development was applied and the impact of several state-of-the-art stationary phase column chemistries (50 × 2.1 mm, sub-2 μm, and core–shell type materials) on the achievable selectivity and resolution was investigated. The work was performed according to quality by design principles using design of experiment with three experimental factors; namely the gradient time (t G), temperature (T), and mobile phase pH. Thanks to modeling software, it was proved that the separation of all compounds was feasible on numerous column chemistries within <10 min, by proper adjustments of variables. It was also demonstrated that the reliability of predictions was good, as the predicted retention times and resolutions were in good agreement with the experimental ones. The final, optimized method separates 16 peaks related to amlodipine and bisoprolol within 7 min, ensuring baseline resolution between all peak-pairs.
    No preview · Article · Sep 2014 · Chromatographia
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    ABSTRACT: Superficially porous particles (SPP), or core shell particles, which consist of a non-porous silica core surrounded by a thin shell of porous silica, have gained popularity as a solid support for chromatography over the last decade. In the present study, five unbonded silica, one diol, and two ethylpyridine (2-ethyl and 4-ethyl) SPP columns were evaluated under SFC conditions using two mixtures, one with 17 drug-like compounds and the other one with 7 drug-like basic compounds. Three of the SPP phases, SunShell™ 2-ethylpyridine (2-EP), Poroshell™ HILIC, and Ascentis(®) Express HILIC, exhibited superior performances relative to the others (reduced theoretical plate height (hmin) values of 1.9-2.5 for neutral compounds). When accounting for both achievable plate count and permeability of the support using kinetic plot evaluation, the Cortecs™ HILIC 1.6μm and Ascentis(®) Express HILIC 2.7μm phases were found to be the best choices among tested SPPs to reach efficiencies up to 30,000 plates in the minimum amount of time. For desired efficiencies ranging from 30,000 to 60,000 plates, the SunShell™ 2-EP 2.6μm column clearly outperformed all other SPPs. With the addition of a mobile phase additive such as 10mM ammonium formate, which was required to elute the basic components with sharp peaks, the Poroshell™ HILIC, SunShell™ Diol and SunShell™ 2-EP phases represent the most orthogonal SPP columns with the highest peak capacities. This study demonstrates the obvious benefits of using columns packed with SPP on current SFC instrumentation.
    Full-text · Article · Aug 2014 · Journal of Chromatography A
  • Szabolcs Fekete · Jenő Fekete · Davy Guillarme
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    ABSTRACT: In this study, the retention changes induced by frictional heating were evaluated for model small compounds (150-190Da) and a small protein, namely insulin (5.7kDa). For this purpose, the effect of longitudinal temperature gradient caused by frictional heating was experimentally dissociated from the combined effect of pressure and frictional heating, by working either in constant and variable inlet pressure modes. Various columns packed with core-shell and fully porous sub-2μm particles were tested. It appears that frictional heating was less pronounced on the column packed with smallest core-shell particles (1.3μm), compared to the ones packed with core-shell and fully porous particles of 1.7-1.8μm. This observation was attributed to the low permeability of this material and the fact that it can only be employed in a restricted flow rate range, thus limiting the generated heat power. In addition, the thermal conductivity of the solid silica core of superficially porous particles (1.4W/m/K) is known to be much larger than that of fully porous silica. Then, the heat dissipation is improved. However, if systems with higher pressure capability would be available and the mechanical stability of 1.3μm core-shell material was extended to e.g. 2000bar, the retention would be more severely impacted. At 2000bar, ∼4.4W heat power and +30°C increase at column outlet temperature is expected. Last but not least, when analyzing large molecules, the impact of pressure overcomes the frictional heating effects. This was demonstrated in this study with insulin (∼5.7kDa).
    No preview · Article · Jul 2014 · Journal of Chromatography A
  • Michael W. Dong · Szabolcs Fekete · Davy Guillarme
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    ABSTRACT: Columns packed with superficially porous particles (SPPs) have created considerable excitement over the last few years. Indeed, this column technology manifests the advantages of fully porous material (loading capacity, retention) and some beneficial properties of nonporous particles (kinetic performance). This review provides an updated overview of the theory behind the success of SPP technology, trends, benefits, and limitations. It also summarizes the latest developments of sub-2-μm SPPs and instrumental constraints associated with their use. Finally, it describes several applications to illustrate the performance and the universal applicability of these newly engineered particles.
    No preview · Article · Jun 2014 · Lc Gc North America
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    Szabolcs Fekete · Alain Beck · Jean-Luc Veuthey · Davy Guillarme
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    ABSTRACT: Size exclusion chromatography (SEC) is a historical technique widely employed for the detailed characterization of therapeutic proteins and can be considered as a reference and powerful technique for the qualitative and quantitative evaluation of aggregates. The main advantage of this approach is the mild mobile phase conditions that permit the characterization of proteins with minimal impact on the conformational structure and local environment. Despite the fact that the chromatographic behavior and peak shape are hardly predictable in SEC, some generic rules can be applied for SEC method development, which are described in this review. During recent years, some improvements were introduced to conventional SEC that will also be discussed. Of these new SEC characteristics, we discuss (i) the commercialization of shorter and narrower columns packed with reduced particle sizes allowing an improvement in the resolution and throughput; (ii) the possibility of combining SEC with various detectors, including refractive index (RI), ultraviolet (UV), multi-angle laser light scattering (MALLS) and viscometer (IV), for extensive characterization of protein samples and (iii) the possibility of hyphenating SEC with mass spectrometry (MS) detectors using an adapted mobile phase containing a small proportion of organic modifiers and ion-pairing reagents.
    Full-text · Article · Apr 2014 · Journal of pharmaceutical and biomedical analysis
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    Balázs Bobály · Davy Guillarme · Szabolcs Fekete
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    ABSTRACT: The aim of this study was to evaluate the possibilities/limitations of recent RP-LC columns packed with 1.6 μm superficially porous particles (Waters Cortecs) and to compare its potential to other existing sub-2 μm core-shell packings. The kinetic performance of Kinetex 1.3 μm, Kinetex 1.7 μm and Cortecs 1.6 μm stationary phases was assessed. It was found that the Kinetex 1.3 μm phase outperforms its counterparts for ultra-fast separations. Conversely, the Cortecs 1.6 μm packing seemed to be the best stationary phase for assays with longer analysis time in isocratic and gradient modes, considering small molecules and peptides as test probes. This exceptional behaviour was attributed to its favourable permeability and somewhat higher mechanical stability (ΔPmax of 1200 bar). The loading capacity of these three columns was also investigated with basic and neutral drugs analyzed under acidic conditions. It appears that the loading capacities of Cortecs 1.6 μm and Kinetex 1.7 μm were very close, while it was reduced by 2-7-fold on the Kinetex 1.3 μm packing. However, this observation is dependent on the nature of the compound and certainly also on mobile phase conditions. This article is protected by copyright. All rights reserved.
    Full-text · Article · Feb 2014 · Journal of Separation Science
  • Róbert Kormány · Jenő Fekete · Davy Guillarme · Szabolcs Fekete
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    ABSTRACT: In this contribution, the possibility to automatically transfer RPLC methods between different column dimensions and instruments was evaluated using commercial modelling software. The method transfer reliability was tested with loratadine and its 7 related pharmacopeial impurities. In this study, state-of-the-art columns packed with superficially porous particles of 5, 2.6, 1.7 and 1.3μm particles were exclusively employed. A fast baseline separation of loratadine and related impurities (Rs,min=2.49) was achieved under the best analytical conditions (i.e. column of 50mm×2.1mm, 1.3μm, 10-90% ACN in 5min, T=40°C, pH=3, F=0.5ml/min). This optimal method was successfully tested on columns packed with other particle sizes, namely 1.7 and 2.6μm, to reduce pressure drop. The selectivities and retentions remained identical, while the peak widths were logically wider, leading to a reduction of peak capacity from 203 to 181 and 159 on the 1.3, 1.7 and 2.6μm particles, respectively. On the minimum, the resolution was equal to 1.54 on the 50mm×2.1mm, 2.6μm stationary phase. Next to this, the method was transferred to columns of different lengths, inner diameters and particle sizes (100mm×3mm, 2.6μm or 150mm×4.6mm, 5μm). These columns were used on other LC instruments possessing larger dwell volumes. The modelling software employed for developing the original method was able to calculate the new gradient conditions to be used. The accuracy of prediction was excellent, as the average retention time errors between predicted and observed chromatograms were -0.11% and 0.45% when transferring the method to 100mm×3mm and 150mm×4.6mm columns, respectively. This work proves the usefulness and validity of HPLC modelling software for transferring methods between different instruments, column dimensions and/or flow rates.
    No preview · Article · Jan 2014 · Journal of pharmaceutical and biomedical analysis
  • Róbert Kormány · Jenő Fekete · Davy Guillarme · Szabolcs Fekete

    No preview · Article · Jan 2014
  • Róbert Kormány · Jenő Fekete · Davy Guillarme · Szabolcs Fekete
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    ABSTRACT: The goal of this study was to evaluate the accuracy of simulated robustness testing using commercial modelling software (DryLab) and state-of-the-art stationary phases. For this purpose, a mixture of amlodipine and its seven related impurities was analyzed on short narrow bore columns (50×2.1mm, packed with sub-2μm particles) providing short analysis times. The performance of commercial modelling software for robustness testing was systematically compared to experimental measurements and DoE based predictions. We have demonstrated that the reliability of predictions was good, since the predicted retention times and resolutions were in good agreement with the experimental ones at the edges of the design space. In average, the retention time relative errors were <1.0%, while the predicted critical resolution errors were comprised between 6.9 and 17.2%. Because the simulated robustness testing requires significantly less experimental work than the DoE based predictions, we think that robustness could now be investigated in the early stage of method development. Moreover, the column interchangeability, which is also an important part of robustness testing, was investigated considering five different C8 and C18 columns packed with sub-2μm particles. Again, thanks to modelling software, we proved that the separation was feasible on all columns within the same analysis time (less than 4min), by proper adjustments of variables.
    No preview · Article · Nov 2013 · Journal of pharmaceutical and biomedical analysis
  • Szabolcs Fekete · Davy Guillarme
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    ABSTRACT: The aim of this work was to evaluate the practical possibilities in gradient elution mode of a column packed with 1.3μm core-shell particles recently released on the market. For this purpose, two types of analytes possessing different diffusion coefficients were selected (small molecule and peptide). It appears that the new 1.3μm material was particularly well suited for fast separations, compared to other existing core-shell particle dimensions in gradient mode. The new material systematically outperforms the other existing ones for peak capacity up to 300 for small molecules and 700 (corresponding to t0=15min) for peptides. Based on these cut-off values, the advantage of column packed with 1.3μm was much more obvious for peptides vs. small molecules analysis. Further improvements in terms of column mechanical stability and system upper pressure capability could expand the limits of separation speed and efficiency to a different level. Again, because of the current pressure limitation and low permeability, a column length of more than 5-8cm is never desired for small molecules analysis in gradient elution. On the contrary, longer columns were useful for peptide analysis. As example, a column of 28cm packed with 1.3μm particles provides a peak capacity of 1000 in the case of peptides analysis. All the predicted values were experimentally confirmed using a standardized extract of Ginkgo biloba and a tryptic digest of a monoclonal antibody (Panitumumab). For the plant extract, the better performance was always achieved with a 5cm long column (P=267 and 268 for the 5 and 15cm, respectively, using a gradient time of 10 and 40min, respectively). Finally, in the case of peptide mapping, a 15cm long column packed with 1.3μm particles was the best choice (P=176 and 311 for the 5 and 15cm, respectively, using a gradient time of 10 and 40min, respectively).
    No preview · Article · Oct 2013 · Journal of Chromatography A
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    ABSTRACT: Recently, there has been a renewed interest in supercritical fluid chromatography (SFC), due to the introduction of state-of-the-art instruments and dedicated columns packed with small particles. However, the achievable kinetic performance and practical possibilities of such modern SFC instruments and columns has not been described in details until now. The goal of the present contribution was to provide some information about the optimal column dimensions (i.e. length, diameter and particle size) suitable for such state-of the-art systems, with respect to extra-column band broadening and system upper pressure limit. In addition, the reliability of the kinetic plot methodology, successfully applied in RPLC, was also evaluated under SFC conditions. Taking into account the system variance, measured at ∼85μL(2), on modern SFC instruments, a column of 3mm I.D. was ideally suited for the current technology, as the loss in efficiency remained reasonable (i.e. less than 10% decrease for k>6). Conversely, these systems struggle with 2.1mm I.D. columns (55% loss in N for k=5). Regarding particle size, columns packed with 5μm particles provided unexpectedly high minimum reduced plate height values (hmin=3.0-3.4), while the 3.5 and 1.7μm packing provided lower reduced plate heights hmin=2.2-2.4 and hmin=2.7-3.2, respectively. Considering the system upper pressure limit, it appears that columns packed with 1.7μm particles give the lowest analysis time for efficiencies up to 40,000-60,000 plates, if the mobile phase composition is in the range of 2-19% MeOH. The 3.5μm particles were attractive for higher efficiencies, particularly when the modifier percentage was above 20%, while 5μm was never kinetically relevant with modern SFC instruments, due to an obvious limitation in terms of upper flow rate value. The present work also confirms that the kinetic plot methodology could be successfully applied to SFC, without the need for isopycnic measurements, as the difference in plate count between predicted and experimental values obtained by coupling several columns in series (up to 400mm) was on average equal to 3-6% and with a maximum of 13%.
    Full-text · Article · Sep 2013 · Journal of Chromatography A
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    ABSTRACT: Innovative columns made with very small core-shell particles (1.0-1.4μm) were investigated over a wide experimental space using state-of-the-art ultra high performance liquid chromatography (UHPLC) instruments. Among the columns tested is one that is now commercially available and is made with 1.3μm core-shell particles consisting of non-porous cores about 0.9μm in size and porous shells <0.2μm thick. This work demonstrated that exceptionally low observed minimum plate heights of 2.2μm could be obtained using columns packed with 1.3μm particles, corresponding to a plate count of over 450,000 plates/m. It was shown that only low volume columns allow operation under optimal conditions with current top-of-the-line UHPLC instruments. It was also demonstrated that the loss in performance caused by frictional heating effects remains negligible. Finally, the practical utility of these columns was confirmed with several real-world applications requiring extreme resolving power (i.e. peptide mapping, sample typical of metabolomic studies and crude human insulin). The performance achieved was compared to that of a reference UHPLC column packed with 1.7μm fully porous particles. The column packed with 1.3μm particles gave peak capacity values that were 20-40% higher than the reference column for the same analysis time.
    No preview · Article · Aug 2013 · Journal of Chromatography A
  • Szabolcs Fekete · Krisztián Horváth · Davy Guillarme
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    ABSTRACT: In this study, pressure induced changes in retention were measured for model peptides possessing molecular weights between ∼1 and ∼4kDa. The goal of the present work was to evaluate if such changes were only attributed to the variation of molar volume and if they could be estimated prior to the experiments, using theoretical models. Restrictor tubing was employed to generate pressures up to 1000bar and experiments were conducted for mobile phase temperatures comprised between 30 and 80°C. As expected, the retention increases significantly with pressure, up to 200% for glucagon at around 1000bar compared to ∼100bar. The obtained data were fitted with a theoretical model and the determination coefficients were excellent (r(2)>0.9992) for the peptides at various temperatures. On the other hand, the pressure induced change in retention was found to be temperature dependent and was more pronounced at 30°C vs. 60 or 80°C. Finally, using the proposed model, it was possible to easily estimate the pressure induced increase in retention for any peptide and mobile phase temperature. This allows to easily estimating the expected change in retention, when increasing the column length under UHPLC conditions.
    No preview · Article · Aug 2013 · Journal of Chromatography A
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    ABSTRACT: When performing fast LC with 50mm narrow-bore columns packed with small particles, the LC instrumentation can give rise to non-negligible band broadening. In the present study, the loss in chromatographic efficiency attributed to nine different mass spectrometers of various brands, ionization source geometries and types of analyzers was assessed. In their standard configurations, the extra-column variance of these UHPLC-MS systems was estimated to vary from 20 to >100μL(2). However, it was demonstrated that these differences arise exclusively from the chromatographic system (i.e., injector, tubing, valves, heater) and from the tubing employed to interface the UHPLC instrument with the MS device. By minimizing the tubing used for each UHPLC system, the extra-column variance was reduced to approximately 17-19μL(2) at 600μL/min, for all types of configurations. To achieve optimal chromatographic performance, it is therefore of prime importance to optimize the UHPLC configuration prior to conducting MS. The tubing located between the UHPLC system and the ionization source entrance was found to be particularly critical, as it contributes to band broadening even in the gradient mode. Using an optimized UHPLC-MS configuration, the loss in efficiency with a 50×2.1mm I.D. column was negligible for k>7. However, the efficiency loss with 1mm I.D. columns remained non-negligible for all current instrumentation, even for solutes with a value of k>20. Indeed, for a mixture of isobaric substrates and metabolites analyzed in gradient mode, the peak widths decreased by approximately 50% between a standard and optimized UHPLC-MS configuration, considering a 50×2.1mm, 1.7μm column. The peak broadening was changed by 230% on a 50×1mm, 1.7μm stationary phase, for the same system configurations.
    Full-text · Article · Aug 2013 · Journal of Chromatography A
  • Szabolcs Fekete · Davy Guillarme
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    ABSTRACT: The goal of this study was to critically evaluate a new generation of columns packed with 1.3μm core-shell particles. The practical possibilities and limitations of this column technology were assessed and performance was compared with other reference columns packed with 1.7, 2.6 and 5μm core-shell particles. The column efficiency achieved with 1.3μm core-shell particles was indeed impressive, Hmin value of only 1.95μm was achieved, this would correspond to an efficiency of more than 500,000plates/m. The separation impedance of this column was particularly low, Emin=2000, mostly due to a reduced plate height, h of 1.50. Comparing the kinetic performance of 1.3μm core-shell particles to that of other particle dimensions tested in this study revealed that the 1.3μm material could provide systematically the shortest analysis time in a range of below 30,000 theoretical plates (N<30,000).Despite its excellent chromatographic performance, it was evident that this column suffers from the limitations of current instrumentation in terms of upper pressure limit and extra-column band broadening: (1) even at 1200bar, it was not possible to reach an optimal linear velocity showing minimal plate height value, due to the low permeability of this column (Kv=1.7×10(-11)cm(2)), and (2) for these short narrow bore columns packed with 1.3μm core shell particles, which is mandatory for performing fast-analysis and preventing the influence of frictional heat on column performance in UHPLC, it was observed that the extra-column band broadening could have a major impact on the apparent kinetic performance. In the present work, significant plate count loss was noticed for retention factors of less than 5, even with the best system on the market (σ(2)ec=2μL(2)).
    No preview · Article · Aug 2013 · Journal of Chromatography A
  • Szabolcs Fekete · Jenő Fekete
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    ABSTRACT: This chapter provides an insight about the theory to what is behind the success of shell particles and presents a summary of the latest applications from different fields. Shell particles are made of a solid, nonporous core surrounded by a shell of a porous material that has properties similar to those of the fully porous materials conventionally used in high performance liquid chromatography (HPLC). Different plate height models are written as the sum of different contributions to peak broadening such as (1) longitudinal diffusion, (2) eddy dispersion, (3) the external film mass transfer, (4) the transparticle mass transfer resistance, and currently (5) the frictional heat term. The chapter reviews the latest applications of shell columns in the field of pharmaceutical, bio, environmental, and food analysis.
    No preview · Chapter · Aug 2013
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    ABSTRACT: In the present study, three types of silica-based monoliths, i.e., the first and second generations of commercial silica monolithic columns and a wide-pore prototype monolith were compared for the analysis of large biomolecules. These molecules possess molecular weights between 1 and 66 kDa. The gradient kinetic performance of the first-generation monolith was lower than that of the second generation, for large biomolecules (>14 kDa) but very close with smaller ones (1.3-5.8 kDa). In contrast, the wide-pore prototype column was particularly attractive with proteins larger than 19 kDa (higher peak capacity). Among these three columns, the selectivity and retention remained quite similar but a possible larger number of accessible and charged residual silanols was noticed on the wide-pore prototype material, which lead to unpredicted small changes in selectivity and slightly broader peaks than expected. The peak shapes attained with the addition of 0.1% formic acid in the mobile phase remained acceptable for MS coupling, particularly for biomolecules of less than 6 kDa. It was found that one of the major issues with all of these silica-based monoliths is the possible poor recovery of large biomolecules (principally with monoclonal antibody fragments of more than 25 kDa). This article is protected by copyright. All rights reserved.
    Full-text · Article · Jul 2013 · Journal of Separation Science

Publication Stats

1k Citations
212.17 Total Impact Points

Institutions

  • 2012-2016
    • University of Lausanne
      • School of Pharmaceutical Sciences (EPGL)
      Lausanne, Vaud, Switzerland
  • 2012-2015
    • University of Geneva
      Genève, Geneva, Switzerland
  • 2008-2014
    • Gedeon Richter Plc
      Budapeŝto, Budapest, Hungary
  • 2011-2013
    • Budapest University of Technology and Economics
      • Department of Inorganic and Analytical Chemistry
      Budapeŝto, Budapest, Hungary