Systematic comparison of a new generation of columns packed with sub‐2 μm superficially porous particles

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 (ΔP_max of 1200 bar). The loading capacity of these three columns was also investigated with basic and neutral drugs analysed 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.     

Investigation of mass transfer properties and kinetic performance of high‐efficiency columns packed with C18 sub‐2 μm fully and superficially porous particles

Three columns packed with 2.0 μm superficially porous particles, 1.7 μm fully porous particles, and monodisperse 1.9 μm fully porous particles with narrow particle size distribution have been deeply characterized from a kinetic point of view. The 1.9 μm column showed excellent kinetic performance, comparable to that of the superficially porous one. These two columns also exhibit flatter c‐branches of the van Deemter curve compared to the 1.7 μm fully porous particles column, resulting in smaller loss of efficiency when they are operated at higher flow rates than the optimal ones. The independent evaluation of each contribution to band broadening has revealed that the difference in kinetic performance comes from the very small eddy dispersion contribution on the 1.9 μm column, surprisingly even lower than that of the superficially porous one. This finding suggests a very good packing of the monodisperse 1.9 μm column. On the other hand, the potential of 1.7 μm fully porous particles is completely broken down by the strong frictional heating effect already arising at relatively low flow rates.     

Theoretical investigation of diffusion along columns packed with fully and superficially porous particles

Chromatographic columns packed with shell particles are now nearly twice more efficient than columns packed with conventional, fully porous particles. Shell particles are made of a solid core surrounded by a porous shell of constant thickness. Diffusion through the bed of packed columns is complex due to their heterogeneity. It involves diffusion through the external and the internal fluid, and surface diffusion. Six diffusion models are compared that combine these diffusion mechanisms. They involve the external porosity of the bed (?(e)), the ratio of the core to the particle diameters (ρ), and the ratio of the shell diffusivity to the bulk diffusion coefficient (Ω). Four different theoretical approaches were considered. They are based on (1) the additivity of the mass flux densities modulated by the obstruction factors caused by non-porous spherical inclusions; (2) the effective medium theory of Landauer; (3) the effective medium theory of Garnett for spherical inclusions; and (4) the probabilistic theory of Torquato (for binary composite materials only). The two Landauer models fail because they cannot account for the obstruction factor imposed by the presence of non-porous spherical inclusions. The ternary Garnett model (3) provides an excellent approximation of the actual diffusion mechanism but the most physically relevant model seems to be the one derived from a combination of the Garnett model for a binary core-shell particle and of the Torquato model for random dispersion of contacting spheres in a matrix. Accurate measurements of axial dispersion coefficients are needed to validate or reject the semi-empirical parallel diffusion models and to select the most appropriate one. The results of such measurements made with the peak parking method for various compounds are reported in the companion paper.     

Rapid determination of parabens in seafood sauces by high‐performance liquid chromatography: A practical comparison of core–shell particles and sub‐2 μm fully porous particles

In this work, the chromatographic performance of superficially porous particles (Halo core–shell C₁₈ column, 50 mm × 2.1 mm, 2.7 μm) was compared with that of sub‐2 μm fully porous particles (Acquity BEH C₁₈, 50 mm × 2.1 mm, 1.7 μm). Four parabens, methylparaben, ethylparaben, propylparaben, and butylparaben, were used as representative compounds for calculating the plate heights in a wide flow rate range and analyzed on the basis of the Van Deemter and Knox equations. Theoretical Poppe plots were constructed for each column to compare their kinetic performance. Both phases gave similar minimum plate heights when using nonreduced coordinates. Meanwhile, the flat C‐term of the core–shell column provided the possibilities for applying high flow rates without significant loss in efficiency. The low backpressure of core–shell particles allowed this kind of column, especially compatible with conventional high‐performance liquid chromatography systems. Based on these factors, a simple high‐performance liquid chromatography method was established and validated for the determination of parabens in various seafood sauces using the Halo core–shell C₁₈ column for separation.     

Combination of a sub‐3 μm superficially porous particle packed column with charged aerosol detection for the simple and sensitive measurement of nine macrolides in human urine

Due to the lack of chromophores in many macrolides, analytical methods based on mass spectrometry and electrochemical detection coupled to liquid chromatography have been suggested to be suitable for the quantification of macrolides in complex matrices. In this study, a simple and sensitive analytical method was established for the simultaneous measurement of nine macrolides in human urine by combining a sub‐3 μm superficially porous particle packed column with charged aerosol detection. After thorough investigation of various sample preparation methods, including two liquid–liquid extraction methods and four solid‐phase extraction methods, HLB solid‐phase extraction was selected and further optimized. Absolute recovery of the optimized sample preparation method ranged from 99.5–110.2%, indicating its very high extraction/clean‐up efficiency. For chromatography, parameters influencing macrolide separation were systematically optimized, and the resulting conditions allowed baseline separation of nine macrolides within 24 min using a very simple mobile phase. The established method was validated for linearity, limit of detection, limit of quantification, absolute recovery, and precision. Based on its limit of detection (0.025–0.100 μg/mL), the method had similar or greater sensitivity than most methods based on electrochemical detection. It was found that the current method was appropriate for application to real human urine samples after drug administration.     

Synthesis and characterization of nonaqueous dispersion particles with photolabile α‐heptadecylphenacyl ester stabilizer chains

We describe a new method for the synthesis of core–shell photolabile nanoparticles. The synthesis begins with the batch emulsion copolymerization of n‐butyl methacrylate (BMA) and ethylene glycol dimethacrylate to form small (20‐nm‐diameter) crosslinked particles with a narrow size distribution. These seeds are then used for a second‐stage emulsion copolymerizations in which BMA and various polar monomers, including methacrylic acid, are added under monomer‐starved conditions. After characterization of the particles, they are transferred to an N,N‐dimethylformamide solution. The cesium salt of the carboxylic acid groups is reacted with 2‐bromo‐1‐phenyl‐octadecan‐1‐one to convert various fractions of the ? COOH groups to the corresponding 2‐benzoylheptadecyl ester groups. These aliphatic ester groups render the surface sufficiently hydrophobic that the particles can be dispersed in common aliphatic hydrocarbons solvents to yield colloidal dispersions, sterically stabilized by the dangling aliphatic chains. Ester groups with a phenyl ketone attached to the β‐carbon are photolabile. Irradiation of the particles with UV light detaches the sterically stabilizing chains from the particle and transforms the surface groups back to COOH groups. This leads to flocculation of the particles. The emphasis in this article is on the optimization of the particle synthesis and the characterization of the particles obtained. © 2001 John Wiley & Sons, Inc. J Polym Sci Part A: Polym Chem 39: 2642–2657, 2001     

Green ultra‐fast high‐performance liquid chromatographic method using a short narrow‐bore column packed with fully porous sub‐2 μm particles for the simultaneous determination of selected pharmaceuticals as surface water and wastewater pollutants

Fast separations are very desirable in laboratories that analyze large numbers of samples per day or those needing short turn‐around times. Traditional HPLC methods using conventional stationary phases and standard column dimensions require significant amounts of organic solvents and generate large volumes of waste. With growing awareness about the environment, the development of green technologies has been receiving increasing attention. In this work, a very fast green analytical method based on LC‐UV using a short narrow bore column packed with fully porous sub‐2 μm particles has been developed for simultaneous determination of nine pharmaceuticals in wastewater and surface water. The chromatographic separation was optimized in order to achieve short analysis time and good resolution for all analytes in a single run. All analytes could be separated in 1 min with good resolution. Sample preparation was executed by solid phase extraction using Oasis HLB cartridges. The method developed was validated based on parameters such as linearity, precision, accuracy, detection, and quantification limits. The recovery ranged from 70.9 to 92.5% with SDs not higher than 5.4%, except for acetaminophen and sulphanilamide. LODs ranged from 0.6–2.5 μg/L, while the LOQs were in the range 2–8 μg/L.     

Simultaneous determination of quinolones in fish by liquid chromatography coupled with fluorescence detection: Comparison of sub‐2 μm particles and conventional C18 columns

A simple and effective multi‐residue analysis method is presented for the extraction and determination of eleven quinolones (pipemidic acid, enoxacin, norfloxacin, ciprofloxacin, lomefloxacin, enrofloxacin, gatifloxacin, difloxacin, oxolinic acid, nalidixic acid and flumequine) in fish tissues. In this study, multi‐residue separations on four columns packed with 5 μm or sub‐2 μm particles were simultaneously developed for the purpose of comparison. Various gradients were optimized and best resolutions were achieved on each column. A short and sub‐2 μm particle‐sized HPLC column was chosen for its advantages in analysis time and column performance. Additionally, considering the matrix effect of the complex crude fish tissue, an effective extraction protocol was also established for sample pre‐treatment procedure. Good recoveries (71–98%) were obtained from samples fortified with a mix of eleven quinolones at three levels, with satisfactory relative standard deviations and limits of detection. As a result, the sub‐2 μm HPLC column and proposed analytical procedures have been evaluated and applied to the analysis of different fish tissues. Detectable residues were observed in 8 of 30 samples, at concentrations ranging from 4.74 to 23.27 μg/kg.     

A combined tryptic peptide and winged peptide internal standard approach for the determination of α‐lactalbumin in dairy products by ultra high performance liquid chromatography with tandem mass spectrometry

A robust ultra high performance liquid chromatography with tandem mass spectrometry method at peptide level was established for measuring α‐lactalbumin in various dairy products. An isotope‐labeled winged peptide (VKKILDKVG*I NYW*L AHKALCSEKL) with extra amino acids of the sequence of signature peptide concatenated at each end as the internal standard was spiked in samples to participate in the whole tryptic digestion process. The peptide VG*I NYW*L AHK that resulted from the isotope‐labeled winged peptide was used as the final isotopically labeled internal standard of the α‐lactalbumin signature peptide (VGINYWLAHK) during the quantitative analysis. The contents of α‐lactalbumin in samples were calculated based on the equimolar relationship between the α‐lactalbumin protein and signature peptide. The optimized molar ratio of trypsin to protein (1:60) and enzymatic digestion time (5 h) could not only improve the digestion efficiency and reduce the cost, but also minimize the period of sample pretreatment. Considering the robustness of the current method using the isotopically labeled internal standard and acceptable measurement cost, its application may promote the development of nutrient investigation and quality control of α‐lactalbumin in dairy products. This protein analysis method might provide a new reference strategy for food analysis and quantitative protein analysis.     

Comparison of a new high‐resolution monolithic column with core‐shell and fully porous columns for the analysis of retinol and α‐tocopherol in human serum and breast milk by ultra‐high‐performance liquid chromatography†

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.