[Show abstract][Hide abstract] 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.
Journal of Chromatography A 08/2014; 1360. DOI:10.1016/j.chroma.2014.07.078 · 4.17 Impact Factor
[Show abstract][Hide abstract] 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).
Journal of Chromatography A 07/2014; 1359. DOI:10.1016/j.chroma.2014.07.030 · 4.17 Impact Factor
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
Journal of pharmaceutical and biomedical analysis 04/2014; 101. DOI:10.1016/j.jpba.2014.04.011 · 2.98 Impact Factor
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
Journal of pharmaceutical and biomedical analysis 01/2014; 94C:188-195. DOI:10.1016/j.jpba.2014.01.037 · 2.98 Impact Factor
[Show abstract][Hide abstract] 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.
Journal of pharmaceutical and biomedical analysis 11/2013; 89C:67-75. DOI:10.1016/j.jpba.2013.10.029 · 2.98 Impact Factor
[Show abstract][Hide abstract] 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).
Journal of Chromatography A 10/2013; 1320. DOI:10.1016/j.chroma.2013.10.061 · 4.17 Impact Factor
[Show abstract][Hide abstract] 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%.
Journal of Chromatography A 09/2013; 1314. DOI:10.1016/j.chroma.2013.09.039 · 4.17 Impact Factor
[Show abstract][Hide abstract] 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.
Journal of Chromatography A 08/2013; 1311. DOI:10.1016/j.chroma.2013.08.065 · 4.17 Impact Factor
[Show abstract][Hide abstract] 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.
Journal of Chromatography A 08/2013; 1311. DOI:10.1016/j.chroma.2013.08.045 · 4.17 Impact Factor
[Show abstract][Hide abstract] 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.
Journal of Chromatography A 08/2013; DOI:10.1016/j.chroma.2013.08.001 · 4.17 Impact Factor
[Show abstract][Hide abstract] 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)).
Journal of Chromatography A 08/2013; 1308. DOI:10.1016/j.chroma.2013.08.008 · 4.17 Impact Factor
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] ABSTRACT: Monoclonal antibodies (mAbs) represent one of the fastest growing areas of new drug development. However, their analytical characterization is complex and generally requires an array of orthogonal analytical techniques. Reversed phase liquid chromatography is a valuable strategy due to its high resolving power and straightforward compatibility to mass spectrometry. The present study demonstrates that high peak capacity can be attained with intact mAb of ∼150kDa, reduced mAb fragments of ∼25-50kDa and also digested mAb generating numerous peptides of ∼0.5-3kDa. Several long columns packed with fully porous wide-pore sub-2μm particles were coupled in series to evaluate the effect of column length on peak capacity. By using three columns of 150mm length, a mobile phase temperature of 80°C and a gradient time of around 20min, peak capacities of 117 and 128 were obtained for a commercial intact mAb and its reduced mAb fragments, respectively. On the other hand, when peptide mapping was performed at 50°C, with a gradient time of 270min and a column length of 450mm, a peak capacity of more than 700 was achieved.
Journal of pharmaceutical and biomedical analysis 05/2013; 83C:273-278. DOI:10.1016/j.jpba.2013.05.022 · 2.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the last decade, an important technical evolution has occurred in pharmaceutical analysis with numerous innovative supports and advanced instruments that have been proposed to achieve fast or ultra-fast separations in LC with an excellent sensitivity and ease of operation. Among the proposed strategies to increase the throughput, the use of short narrow-bore columns packed with sub-3μm core-shell and porous sub-2μm particles have emerged as the gold standards. Nevertheless, to take the full benefits of these modern supports, a suitable chromatographic system has to be employed. This review summarizes the instrumental needs and challenges in terms of extra-column variance, dwell volume, maximum system pressure, detector data acquisition rate, and injection cycle time. In addition, because of their reasonable pressure drop, the use of columns packed with sub-3μm core-shell particles on a conventional LC instrument is discussed in detail. A methodology is proposed to check the compatibility between stationary phase and instrument, and some solutions are proposed to improve the performance of standard instruments. Finally, because the column technology is evolving faster than instrumentation, it is nowadays possible to purchase short, narrow-bore columns packed with 1.3μm core-shell particles. Micro columns (1mm I.D.) packed with 1.7-1.9μm porous particles are also available from several providers, which limit frictional heating effects and reduce solvent and sample consumption. However, it remains difficult to find instruments compatible with such column geometries and a severe loss of performance may be observed due to the system itself.
Journal of pharmaceutical and biomedical analysis 03/2013; 87. DOI:10.1016/j.jpba.2013.03.012 · 2.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This paper presents new reversed phase liquid chromatographic methods (HPLC-FLD and LC-MS/MS) for the quantification of sulfonamides in spiked and incurred honey samples. The sample preparation was optimized using Oasis HLB (hydrophilic–lipophilic balance) solid-phase extraction (SPE) cartridge. Elutions of sulfonamides were carried out under acidic, neutral, and basic conditions using methanol. Recoveries under acid condition were in the range from 66.8–90%, which were approximately 10% higher than those obtained under other conditions. The sample clean-up was also tested using Strata-XL cartridges. The HPLC-FLD separation was performed using a Varian C18 column and a ternary (methanol-acetonitrile-phosphate buffer, pH 5) mobile phase resulting good selectivity for the determination. The robustness of the ternary gradient method was evaluated by computer simulation (DryLab). LC-MS/MS separation was carried out on a Kinetex XB core-shell type HPLC column that enabled a low limit of detection (0.01–0.5 µg/kg) and faster separation (6 min). The developed methods were validated in accordance with the European Union Commission Decision 2002/657/EC and were applied successfully for more than four hundred honey samples (under a national monitoring program). The concentrations of sulfadimethoxine, sulfachloropyridazine, and trimethoprim residues in samples were found in a concentration range from 0.03 up to 686 µg/kg.
Journal of Liquid Chromatography & Related Technologies 03/2013; 36(8). DOI:10.1080/10826076.2012.685911 · 0.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A new size exclusion chromatography column packed with 1.7μm particles and possessing 200Å pore size has been critically evaluated for the determination of proteins and monoclonal antibody aggregates. In a first instance, the kinetic performance of this column was compared with that of a conventional column packed with 5μm particles and with a recently launched 3μm material, also possessing 200Å pore size. In average, 2-5 times lower plate height were achieved on the 1.7μm packing, compared with the conventional 5μm particles. It was also demonstrated that elevated mobile phase temperature (up to 50 or 60°C) allows improving the kinetic efficiency by 20-40% in size exclusion chromatography, compared to 30°C. On the other hand, the new 3μm material performed only slightly lower kinetic efficiency than the 1.7μm one. When considering the upper pressure and temperature limits of these three columns, the 1.7μm column systematically outperforms the 5 and 3μm materials in the "practical" plate number range (N<30,000) and analysis times could be cut by 2-4 times. The column packed with 5μm particles was only beneficial for plate counts beyond 100,000 plates, while the 3μm packing could be considered as a good compromise between speed, efficiency and pressure. Besides the excellent kinetic performance of 1.7μm size exclusion material under high temperature conditions, some artifacts were observed when quantifying protein aggregates. Indeed, both high pressure observed with 1.7μm particles (shear forces, frictional heating) and elevated temperature produce some non negligible amount of on-column additional protein aggregates.
Journal of pharmaceutical and biomedical analysis 02/2013; 78-79C:141-149. DOI:10.1016/j.jpba.2013.02.013 · 2.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Various recent wide-pore reversed-phase stationary phases were studied for the analysis of intact monoclonal antibodies (mAbs) of 150 kDa and their fragments possessing sizes between 25 and 50 kDa. Different types of column technology were evaluated, namely, a prototype silica-based inorganic monolith containing mesopores of ∼250 Å and macropores of ∼ 1.1 μm, a column packed with 3.6 μm wide-pore core-shell particles possessing a wide pore size distribution with an average around 200 Å and a column packed with fully porous 1.7 μm particles having pore size of ∼300 Å. The performance of these wide-pore materials was compared with that of a poly(styrene-divinyl benzene) organic monolithic column, with a macropore size of approximately 1 μm but without mesopores (stagnant pores). A systematic investigation was carried out using model IgG1 and IgG2 mAbs, namely rituximab, panitumumab, and bevacizumab. Firstly, the recoveries of intact and reduced mAbs were compared on the two monolithic phases, and it appeared that adsorption was less pronounced on the organic monolith, probably due to the difference in chemistry (C18 versus phenyl) and the absence of mesopores (stagnant zones). Secondly, the kinetic performance was investigated in gradient elution mode for all columns. For this purpose, peak capacities per meter as well as peak capacities per time unit and per pressure unit (PPT) were calculated at various flow rates, to compare performance of columns with different dimensions. In terms of peak capacity per meter, the core-shell 3.6 μm and fully porous 1.7 μm columns outperformed the two monolithic phases, at a temperature of 60 °C. However, when considering the PPT values, the core-shell 3.6 μm column remained the best phase while the prototype silica-based monoliths became very interesting, mostly due to a very high permeability compared with the organic monolith. Therefore, these core-shell and silica-based monolith provided the fastest achievable separation. Finally, at the maximal working temperature of each column, the core-shell 3.6 μm column was far better than the other one, because it is the only one stable up to 90 °C. Lastly, the loading capacity was also measured on these four different phases. It appeared that the organic monolith was the less interesting and rapidly overloaded, due to the absence of mesopores. On the other hand, the loading capacity of prototype silica-based monolith was indeed reasonable.